Beginning her career on the soap opera One Life to Live, she also had minor roles in Random Hearts, and You've Got Mail. After guest starring roles in Law and Order SVU, Now and Again, and The West Wing, Aylesworth was part of the 2001 series All Souls, which was cancelled during its first season. In 2002, Aylesworth joined the cast of another series, American Embassy which also failed to gain an audience.

In 2001, Aylesworth had auditioned for the part of turncoat agent Nina Myers on 24 but lost to Sarah Clarke. In 2002, she again auditioned for the role of Kate Warner, but lost to Sarah Wynter. The producers, however, asked her to join the cast as recurring character Michelle Dessler, a CTU agent.
Spoiler warning: Plot and/or ending details follow.

Her character quickly developed a relationship with fellow agent Tony Almeida (Carlos Bernard), and became a firm fan favorite.

By the third season, Aylesworth had become a main cast member and, when the writers decided to have major cast changes at the end of that season, including the departure of her character, fan forums lit up with people asking for her to stay. In the twelfth episode of the fourth season, Aylesworth returned - Michelle now a high-ranking bureaucrat in the organization. She remained for the remainder of the fourth season, but the character was killed in the first moments of Season 5.

Aylesworth also had a much-publicized guest starring role on CSI which was initially rumored to be a regular role. However, after fired cast members Jorja Fox and George Eads were rehired, and with the offer to return to 24, Aylesworth's role became just one episode long.