Aylesworth began her career on the soap opera One Life to Live. She also appeared in minor roles in Random Hearts, and You've Got Mail. After guest roles in Law & Order, Law & Order: Special Victims Unit, Now and Again, The West Wing, and Ed, Aylesworth was cast in the 2001 series All Souls, which was canceled during its first season. In 2002, Aylesworth starred in the series The American Embassy — her character replaces one killed during the pilot — that also failed to gain an audience.

In 2001, Aylesworth auditioned for the part of Nina Myers on FOX Network's 24 but lost to Sarah Clarke. In 2002, she auditioned for another role on "24", Kate Warner, and lost to Sarah Wynter. The producers asked her to join the cast as recurring character Michelle Dessler, a CTU agent.

Aylesworth's character, "Michelle Dessler", soon developed a relationship with fellow agent Tony Almeida (Carlos Bernard), and became popular with fans. By the third season, Aylesworth had bec