Glau received a scholarship to a ballet company and was home-schooled from grades 3 to 12 to accommodate her ballet training. Glau was classically trained as a ballet dancer and also studied tango and flamenco. After a broken toe ended her dancing career, Glau later moved to Los Angeles in 2002 to pursue a more active acting career. Glau's first television credit was a guest role as a ballerina on the 2002 episode "Waiting in the Wings" of the television series Angel. There she caught the eye of director Joss Whedon, who later cast her in his critically acclaimed but short-lived TV series Firefly as River Tam, a role she reprised for the show's feature film sequel, Serenity. Glau previously auditioned for the role as a Pink Ranger on one of the Power Rangers series before meeting Joss Whedon.
Glau appeared on the episode "Love Conquers Al" of the TV series Cold Case. She had a small role in the film Sleepover, in which she played a high school senior named Shelly. She also appeared in the CSI episode "What's Eating Gilbert Grissom?", and the second season of The Unit; portraying Crystal Burns, the girlfriend of Jeremy Erhart. In 2006, Glau played Tess Doerner, a paranoid schizophrenic returnee in the second season premiere of The 4400 (episode "Wake-Up Call"), and became a recurring character from season 3 onwards. She also starred in the science fiction film Mammoth. Glau was cast in the ABC Family TV movie The Initiation of Sarah.