4-1BB is expressed on activated CD4+ and CD8+ T cells; its ligand, 4-1BB ligand is expressed on APCs. Despite expression on both T cell subpopulations, 4-1BB has been reported to predominantly affect CD8+ T cell responses. By quantifying graft-vs-host disease alloresponses in vivo, we demonstrate that both CD4+ and CD8+ T cell-mediated alloresponses are regulated by 4-1BB/4-1BB ligand interactions to approximately the same extent. 4-1BB receptor-facilitated CD4+ T cell-mediated alloresponses were partly CD28 independent. In two distinct marrow graft rejection systems, host CD8+ and CD4+ T cells each separately contributed to host anti-donor T cell-mediated allograft rejection. α4-1BB mAb increased the graft-vs-leukemia effect of a suboptimal number of donor splenocytes given later post bone marrow transplantation by bolstering allogeneic responses resulting in leukemia elimination. In summary, 4-1BB ligation is a potent regulator of CD4+ and CD8+ T cell-mediated allogeneic responses in vivo. Modifying the ligation of 4-1BB represents a new approach to altering the graft-vs-host disease and graft-vs-leukemia effects of allogeneic T cells post bone marrow transplantation.