Graft-versus-host disease (GVHD) is a serious complication of bone marrow transplantation that is induced by an alloreactive donor T cell response. Regulatory T cells (Treg) play a crucial role in suppressing effector T cells to limit immune responses. GVHD severity is associated with Treg deficiency, and dysregulated Treg populations are also associated with autoimmune and allergic disease and anti-tumour immunity. Thus, the promotion of Treg homeostasis is a promising strategy to induce tolerance. Autophagy is a self-degradative process for cytosolic components which promotes cell homeostasis and survival. It is characterized by the formation of double membrane structures called autophagosomes, which require coordinated interactions of ATG5, ATG7 and LC3 proteins for their formation. Using Western blot analysis and imaging flow cytometry to quantify LC3 production and aggregation, we demonstrate for the first time that autophagy is an active process in Treg. Furthermore, in chimeric mice with a haematopoietic-restricted ATG5 deficiency, Treg develop normally in the thymus but are dramatically reduced in the periphery compared to wild-type chimeras. Using ATG7flflxFoxP3cre mice in which ATG7 deficiency was restricted to Treg we confirmed this effect was autophagy dependent and Treg intrinsic. Whereas, fully functional ATG5-/- Treg could be efficiently induced in vitro (iTreg), ATG5-/- iTreg failed to survive after transfer into irradiated syngeneic recipients. Thus, although autophagy is not required for central Treg development, it is critical for peripheral Treg homeostasis and survival after transplantation. Our results suggest that promoting autophagy in Treg is an attractive strategy to improve their survival and promote immunological tolerance. In this regard, the use of the immunosuppressant rapamycin, a known inducer of autophagy, may function to control alloimmune responses, at least in part, by specifically promoting Treg survival.