Although CD4(+)CD25(+) T cells (T regulatory cells [Tregs]) and natural killer T cells (NKT cells) each protect against graft-versus-host disease (GVHD), interactions between these 2 regulatory cell populations after allogeneic bone marrow transplantation (BMT) have not been studied. We show that host NKT cells can induce an in vivo expansion of donor Tregs that prevents lethal GVHD in mice after conditioning with fractionated lymphoid irradiation (TLI) and anti-T-cell antibodies, a regimen that models human GVHD-protective nonmyeloablative protocols using TLI and antithymocyte globulin (ATG), followed by allogeneic hematopoietic cell transplantation (HCT). GVHD protection was lost in NKT-cell-deficient Jalpha18(-/-) hosts and interleukin-4 (IL-4)(-/-) hosts, or when the donor transplant was Treg depleted. Add-back of donor Tregs or wild-type host NKT cells restored GVHD protection. Donor Treg proliferation was lost in IL-4(-/-) hosts or when IL-4(-/-) mice were used as the source of NKT cells for adoptive transfer, indicating that host NKT cell augmentation of donor Treg proliferation after TLI/antithymocyte serum is IL-4 dependent. Our results demonstrate that host NKT cells and donor Tregs can act synergistically after BMT, and provide a mechanism by which strategies designed to preserve host regulatory cells can augment in vivo donor Treg expansion to regulate GVHD after allogeneic HCT.