CD4⁺CD25⁺ regulatory T (Treg) cells are potent modulators of alloimmune responses. In murine models of allogeneic bone marrow transplantation, adoptive transfer of donor CD4⁺CD25⁺ Treg cells protects recipient mice from lethal acute graft-versus-host disease (aGVHD) induced by donor CD4⁺CD25^- T cells. Here we examined the differential effect of CD62L⁺ and CD62L^- subsets of CD4⁺CD25⁺ Treg cells on aGVHD-related mortality. Both subpopulations showed the characteristic features of CD4⁺CD25⁺ Treg cells in vitro and did not induce aGVHD in vivo. However, in cotransfer with donor CD4⁺CD25^- T cells, only the CD62L⁺ subset of CD4⁺CD25⁺ Treg cells prevented severe tissue damage to the colon and protected recipients from lethal aGVHD. Early after transplantation, a higher number of donor-type Treg cells accumulated in host mesenteric lymph node (LN) and spleen when CD4⁺CD25⁺CD62L⁺ Treg cells were transferred compared with the CD62L^- subset. Subsequently, CD4⁺CD25⁺CD62L⁺ Treg cells showed a significantly higher capacity than their CD62L^- counterpart to inhibit the expansion of donor CD4⁺CD25^- T cells. The ability of Treg cells to efficiently enter the priming sites of pathogenic allo-reactive T cells appears to be a prerequisite for their protective function in aGVHD.