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Sam Winchester was born on May 2, 1983 to John and Mary Winchester. Sam was the couple's second-born child and younger son, four years younger than his older brother, Dean Winchester. Sam is different from his older brother, Dean, in that he is rational, sensitive, and seemingly more concerned with living a "normal life" than his brother; in spite of this, it is apparent that he would do anything for Dean. He also rebelled against his father's attempts to raise him as a demon hunter and enrolled in Stanford University. When Dean came to ask Sam for his help in the first episode, the viewers are introduced to Jessica, Sam's girlfriend. It is apparent that Sam was somewhat popular, despite no one knowing what it is his family really does. His brother, Dean, is the only one allowed to call him by his nickname, Sammy. Sam has also had more love interests than Dean, although Dean has had a few flings on the show.

Sam Winchester was born on May 2, 1983. When he was only six months old (November 2, 1983), his mother Mary was killed in his nursery by The Demon. Infant Sam was saved when his older brother, Dean, ran outside with him while their father John went back to attempt to rescue Mary, who was stuck on the ceiling and consumed by the flames that later engulfed the entire house.

Twenty two years later to the day, the same demon killed Sam's girlfriend Jessica Moore, which spurs Sam to embark on a journey to find their missing father and get vengeance on the demon. While John Winchester is missing, he occasionally contacts the boys to leave them new missions to complete. The boys fend off mystical creatures and urban legends such as the Woman in White, the wendigo, folklore's Bloody Mary, and shapeshifters. Through these missions, Sam begins experiencing episodes of precognition and once displays telekinesis.

The season finale concludes with Sam, Dean, and their father John escaping from a horrific crash with The Demon in Salvation, Iowa. While Sam is driving the Winchesters to a hospital after Dean and John were hurt, a truck crashes into them, causing massive damage to Dean's Impala with the Winchesters inside.

In the beginning of the second season, Sam takes a more active role in the hunting. He tells Dean that this is what their dad would have wanted him to do. The Demon's plans for Sam are still unknown. Dean and Sam meet with three people that John once knew: Ellen Harvelle, her daughter Jo, and their computer genius associate Ash. They assist the brothers in their hunting. In the episode "Simon Said", Sam displays immunity to Andy Gallagher's power of mind control, as opposed to Dean who gave him his beloved Impala. He is also immune to the demonic sulfur virus in the episode "Croatoan".

Since "Hunted", Sam has learned what his father told Dean moments before his death: that the Demon plans to cause Sam to become evil and use him and "children like him" as soldiers in an upcoming war. Dean was told that if he could not save Sam, he would have to kill him. Sam is convinced that he must save as many people as he can in order to change his destiny, as he drunkenly states in "Playthings".

In the episode "Born Under a Bad Sign", Sam is possessed by the same demon responsible for Meg's possession in Season 1. It is also hinted at that this is also the same demon that lead to the death Ellen's husband. It becomes clear that if the time comes for Dean to shoot his brother, Dean would not be able to do so. In this episode Sam(while still possessed) also kills another hunter.

In the episode "What Is and What Should Never Be", Sam and Dean aren't close in the alternate reality created by a djinn. Neither Sam nor Dean had ever hunted, and Jessica is Sam's fiancee. As in reverse to reality, their mother is alive, but their father is dead.

The first episode of the season finale of season two, All Hell Breaks Loose, Part 1, Sam is trapped in a haunted abandoned town in South Dakota with "Others" of his kind. It was revealed that there is demon blood in Sam and his mother Mary Winchester knew who the demon was, much to Sam's surprise. He is stabbed by a soldier named Jake and collapses in his older brother's arms just as he and fellow hunter Bobby arrive to the rescue. He dies within the minute, amongst his brother's pleas for him to stay with him. In the second episode of the season finale, Dean made a deal with a "Crossroads Demon" to deliver himself to Hell in one year, in exchange for Sam's life. Sam doesn't know of what happened while he was dead, and is in fact unaware he was ever dead in the first place. In the following battle to prevent Jake and the "Yellow-Eyed Demon" from unleashing a demonic army, Sam shoots and kills Jake, unloading seven bullets into his body - 4 in the back and 3 in Jake's face. This is the first time Sam kills a man, or someone that isn't a demon, and seems to show no remorse, this could be because Jake killed him prior.. The "Yellow-Eyed-Demon" questions Dean if he is sure that what he brought back in Sam is 100% pure. Sam figures out that he was dead, and tells Dean he'll get him out of the deal he made, no matter what.

Sam uses a chromed Beretta 92FS loaded with silver rounds and uses a pistol grip 4-shelled 12 gauge pump shotgun loaded with rock salt.

In season one he owns what appears to be a Dell Precision M65 laptop, which he uses to gather information for hunts. The first time he uses the laptop is in the second episode "Wendigo" where he plays a video message. His possession of the laptop is ambiguous at this point because it is unsure what he did and did not salvage from the fire at Stanford in the episode "Pilot". In that same episode, his brother Dean makes references using computer software which leads viewers to believe that Dean may, in fact, be the owner of the laptop. In episode fifteen, "Tall Tales," Sam claims ownership of the laptop, accusing Dean of misplacing it.

In the beginning of season two, when Sam and Bobby are surveying the wreckage of the totaled Impala, Sam pulls the broken pc from the passenger seat. Then, in the second season's eighth episode, "Crossroad Blues," Sam is seen using an Apple 15" PowerBook or MacBook Pro, though interestingly, its display shows a Windows OS.

Sam also appears to own a Verizon-branded Motorola Q which has on at least one occasion (in "Hollywood Babylon") been used to visually track unseen ghosts. This phone however was destroyed in the season 3 episode "Fresh Blood".

Sam exhibits both telekinetic and precognitive abilities at various times throughout the show. His telekinesis onscreen is limited to a single incident wherein he was under extreme stress and pressure, while his precognitive abilities, manifesting as dreams and visions of others' deaths, started prior to the first episode with dreams of Jessica Moore's death, and continued throughout the first two seasons. Sam stated that visions stopped with the death of Azazel at the end of season two, who is assumed to have given Sam and the other psychic children their gifts.

As one of the stars of the series Sam has appeared in all the episodes. However, most episodes are focused on either Sam or Dean.