CD4⁺CD25⁺ immunoregulatory T cells play a pivotal role in preventing organ-specific autoimmune diseases and in tolerance induction to allogeneic organ transplants. We investigated whether these cells could also control graft-versus-host disease (GVHD), the main complication after allogeneic hematopoietic stem cell transplantation (HSCT). Here, we show that the few CD4⁺CD25⁺ T cells naturally present in the transplant regulate GVHD because their removal from the graft dramatically accelerates this disease. Furthermore, the addition of freshly isolated CD4⁺CD25⁺ T cells at time of grafting significantly delays or even prevents GVHD. Ex vivo–expanded CD4⁺CD25⁺ regulatory T cells obtained after stimulation by allogeneic recipient-type antigen-presenting cells can also modulate GVHD. Thus, CD4⁺CD25⁺ regulatory T cells represent a new therapeutic tool for controlling GVHD in allogeneic HSCT. More generally, these results outline the tremendous potential of regulatory T cells as therapeutics.