The use of TGF-β–induced CD4+ Foxp3+ T cells (induced regulatory T cells [iTregs]) is an important prevention and treatment strategy in autoimmune diseases and other disorders. However, the potential use of iTregs as a treatment modality for acute graft-versus-host disease (aGVHD) has not been realized because they may be unstable and less suppressive in this disease. We restudied the ability of iTregs to prevent and treat aGVHD in two mouse models. Our results showed that, as long as an appropriate iTreg-generation protocol is used, these iTregs consistently displayed a potent ability to control aGVHD development and reduce mortality in the aGVHD animal models. iTreg infusion markedly suppressed the engraftment of donor CD8+ cells and CD4+ cells, the expression of granzyme A and B, the cytotoxic effect of donor CD8+ cells, and the production of T cell cytokines in aGVHD. Therefore, we conclude that as long as the correct methods for generating iTregs are used, they can prevent and even treat aGVHD.