Bledel made her TV debut on The WB series Gilmore Girls in 2000. She portrayed Rory Gilmore, the daughter of single mother Lorelai Gilmore. Initially, Rory was a high school student living with her mother, but later moved on to college at Yale University, where she, among other things, worked as the editor of the Yale Daily News. Bledel has said that she feels she can relate in some ways to Rory, both when it comes to some personal traits and her relationship to her own parents. She also sees the family structure and mother-daughter relationship on the show as something to which many people can relate.

In 2002, Bledel was voted one of Teen People's "25 Hottest Stars Under 25". She appeared as the main character in the video for Less Than Jake's "She's Gonna Break Soon", lead single from their 2003 album Anthem. The single charted at number 39 on the UK charts. In 2005, she starred in The Sisterhood of the Traveling Pants based on the book by Ann Brashares. She played Lena Kali