The maintenance of immune homeostasis requires regulatory T cells (T-regs). Given their intrinsic self-reactivity, Tregs must stably maintain a suppressive phenotype to avoid autoimmunity. We report that impaired expression of the transcription factor (TF) Helios by FoxP3(+) CD4 and Qa-1-restricted CD8 T-regs results in defective regulatory activity and autoimmunity in mice. Helios-deficient T-regs develop an unstable phenotype during inflammatory responses characterized by reduced FoxP3 expression and increased effector cytokine expression secondary to diminished activation of the STAT5 pathway. CD8 T-regs also require Helios-dependent STAT5 activation for survival and to prevent terminal T cell differentiation. The definition of Helios as a key transcription factor that stabilizes T-regs in the face of inflammatory responses provides a genetic explanation for a core property of T-regs.