During developerment, Raven Software wanted to include characters from the wider Marvel universe and proposed adding Iron Man or Captain America. Upon hearing this, Marvel persuaded them to use Iron Man. Dan Vondrak, the game's director, believed that Marvel's push for Iron Man was because they wanted to use his inclusion in the game to set up promotion for the 2008 Iron Man movie. Captain America was later added in Marvel: Ultimate Alliance as a playable character.

According to director Dan Vondrak, Deadpool was originally planned to just be an NPC before it was decided to promote him to being a playable character. However, Marvel was initially hesitant about him being playable, as he was considered an obscure and niche character at the time. Meanwhile, Activision conducted focus testing to determine how many people recognized or liked the game's roster. The results for Deadpool in this study saw him being one of the least recognizable yet most beloved characters, which persuaded Marvel to sign off on his usage under the condition that he would only be unlockable after completing the campaign.