Regulatory T cells (Tregs), in particular CD4+ Foxp3+ T cells, have been shown to play an important role in the maintenance of tolerance after allogeneic stem cell transplantation. In the current study, we have identified a population of CD8+ Foxp3+ T cells that are induced early during graft-versus-host disease (GVHD), constitute a significant percentage of the entire Treg population, and are present in all major GVHD target organs. These cells expressed many of the same cell surface molecules as found on CD4+ Tregs and potently suppressed in vitro alloreactive T cell responses. Induction of these cells correlated positively with the degree of MHC disparity between donor and recipient and was significantly greater than that observed for CD4+-induced Tregs (iTregs) in nearly all tissue sites. Mice that lacked the ability to make both CD8+ and CD4+ iTregs had accelerated GVHD mortality compared with animals that were competent to make both iTreg populations. The absence of both iTreg populations was associated with significantly greater expansion of activated donor T cells and increased numbers of CD4+ and CD8+ T cells that secreted IFN-γ and IL-17. The presence of CD8+ iTregs, however, was sufficient to prevent increased GVHD mortality in the complete absence of CD4+ Tregs, indicating at least one functional iTreg population was sufficient to prevent an exacerbation in GVHD severity, and that CD8+ iTregs could compensate for CD4+ iTregs. These studies define a novel population of CD8+ Tregs that play a role in mitigating the severity of GVHD after allogeneic stem cell transplantation.