The show features an X-Men similar in look and line-up to the early 1990s X-Men drawn by Jim Lee, composed of Professor X, Cyclops, Beast, Jean Grey, Wolverine, Rogue, Gambit, Storm and Jubilee, as well as an original character, Morph. Though they were not part of the team as featured in the animated series, the following X-Men have all guest-starred in at least one episode: Colossus, Nightcrawler, Emma Frost, Forge, Havok, Polaris, Cannonball, Banshee, Iceman, Archangel, Psylocke, Cable and Bishop. Keen-eyed fans may also spot cameos by other familiar mutants, such as Feral or Rictor, and Marvel heroes such as Captain America, Ghost Rider and The Punisher.

A number of famous storylines and events from the comics are loosely adapted in the series, such as the Dark Phoenix Saga, Days of Future Past, the Phalanx Covenant, and the Legacy Virus. The third episode, "Enter Magneto", features a confrontation at a missile base: this is largely based on the X-Men's first battle with Magneto, as told in their 1963 debut X-Men #1. The season 4 episodes "Sanctuary, Parts I & II", which involve Magneto creating an orbiting haven for mutants, were influenced by several storylines from the canon, chiefly the first three issues of X-Men (volume 2) and the Fatal Attractions crossover.

Beyond faithfully recreating many of the popular characters and stories from the comic books, the series also dealt fairly openly with mature social issues. The ills of prejudice, intolerance, isolation and racism were all frequent themes in the animated series, as they were in the comics. The series also deals with other social issues, albeit sometimes in subtext, that are not often dealt handled by children's programming: divorce ("Proteus"), Christianity ("Nightcrawler" & "Bloodlines"), the Holocaust ("Enter Magneto", "Deadly Reunions" & "Days of Future Past"), AIDS hysteria ("Time Fugitives"), and even satires of television itself ("Mojovision" and "Longshot"). The Friends of Humanity, a prominent anti-mutant group that appears in the second season, bears great resemblance to the Ku Klux Klan and other white-supremacy groups. A mild touch of transgenderism was introduced as shapeshifting mutants such as Mystique and Morph would often assume both male and female forms. The show, along with Batman: The Animated Series, is considered to be a more adult oriented animated show rather than specifically for children.

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