After allogeneic hematopoietic cell transplantation (HCT), the high inverse correlation between graft-versus-host disease (GVHD) and leukemic relapse requires that calculated measures be taken to reduce GVHD pathology while retaining the graft-versus-leukemia (GVL) effect. We sought to determine whether donor CD4⁺CD25⁺ regulatory T cells could control ongoing GVHD, thereby providing an initial window of time in which the alloreactive anti-host response is permitted to begin, with the intent of most effectively eliminating residual leukemia cells. Prevention of lethal GVHD by infusion of donor CD4⁺CD25⁺ cells early after HCT (day 2) was achieved across a major histocompatibility complex barrier in the haploidentical C3H→(B6xC3H)F₁ model. However, in vitro expansion of donor CD4⁺CD25⁺ T cells, stimulated by recipient cells in the presence of high-dose interleukin-2, was required for successful regulation. In contrast, in the major histocompatibility complex-matched, minor histocompatibility antigen-disparate, CD8-mediated B10.BR→CBA GVHD model, lethal disease could be completely prevented by a single infusion of freshly isolated donor CD4⁺CD25⁺ cells administered as late as 10 days after HCT. Of importance, this late regulatory effect required only a 3:1 ratio of effector CD8:CD4⁺CD25⁺ T cells, indicating a strong potential for the delayed infusion of CD4⁺CD25⁺ cells to control GVHD across minor histocompatibility antigen barriers. Furthermore, this regulation did not interfere with complete and lasting donor engraftment of the hematopoietic compartment. Of most significance, the day 10 infusion of donor CD4⁺CD25⁺ cells into CBA HCT recipients that had been challenged with the MMCBA6 myeloid leukemia cell line did not block an effective GVL response, despite reducing lethal GVHD. These results suggest that donor CD4⁺CD25⁺ T cells infused soon after transplantation can ameliorate the development of GVHD without sacrificing a sufficient GVL effect. © 2003 American Society for Blood and Marrow Transplantation Biology of Blood and Marrow Transplantation 9:243-256 (2003)