Glau was classically trained as a ballerina and also studied tango and flamenco. A professional dancer since age 12, she appeared in several television commercials.

Having relocated to Los Angeles in 2002, Glau's first television credit was a guest role as a ballerina on the 2002 episode "Waiting in the Wings" (episode 3x13) of the television series Angel. There she caught the eye of director Joss Whedon, who later cast her in his short-lived TV series Firefly as River Tam, a role she reprised for the show's feature film sequel, Serenity.

Glau appeared on the episode "Love Conquers Al" (1x06) 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 seas