The proper restraint of the destructive potential of the immune system is essential for maintaining health. Regulatory T (Treg) cells ensure immune homeostasis through their defining ability to suppress the activation and function of other leukocytes. The expression of the transcription factor forkhead box protein P3 (FOXP3) is a well-recognized characteristic of Treg cells, and FOXP3 is centrally involved in the establishment and maintenance of the Treg cell phenotype. In this Review, we summarize how the expression and activity of FOXP3 are regulated across multiple layers by diverse factors. The therapeutic implications of these topics for cancer and autoimmunity are also discussed.