Foxp3, an X chromosome–encoded forkhead transcription factor family member, is indispensable for the differentiation of regulatory T cells. These cells have a vital role in preventing autoimmunity and pathology inflicted by uncontrolled immune responses to infections. Deficiency or mutation in Foxp3 in humans and mice leads to an early onset, highly aggressive and fatal autoimmune disease affecting various tissues. Here, we review recent advances in our understanding of the Foxp3-dependent molecular and functional program and the role of Foxp3 in regulatory T cell biology.