In murine models of allogeneic bone marrow transplantation (BMT), MHC-mismatched recipients given a delayed infusion of donor leukocytes (DLI) at 21 days posttransplant develop significant GVHD whereas MHC-matched recipients do not. The current study was initially designed to test the hypothesis that small numbers of T cells in the MHC-mismatched donor bone marrow (BM) graft exacerbated graft-vs-host disease (GVHD) when DLI was administered at 21 days after BMT. Ex vivo depletion of Thy1+ cells from the donor BM had no impact on the severity of GVHD after DLI. However, depletion of donor T cells in vivo with a Thy1 allele-specific mAb given after BMT resulted in significantly more severe GVHD after DLI. Similar results were obtained in a MHC-matched model of allogeneic BMT, indicating that this was a general phenomenon and not model dependent. These results indicated that a population of donor-derived Thy1+ cells suppressed graft-vs-host reactivity after DLI. Results of experiments with thymectomized recipients demonstrated that an intact thymus was required for generation of the immunoregulatory donor cells. Experiments using TCR β-chain knockout mice as BM donors indicated that the immunosuppressive Thy1+ cells coexpressed αβTCR heterodimers. Similar experiments with CD4 and CD8 knockout donor BM suggested that the immunoregulatory Thy1+ αβTCR+ cells consisted of two subpopulations: a CD4+ CD8− subpopulation and a CD4− CD8− subpopulation. Together, these results show that thymus-derived, Thy1+ αβTCR+ donor cells generated early after allogeneic BMT suppress the graft-vs-host reactivity of T cells given as DLI. These cells may mediate dominant peripheral tolerance after allogeneic BMT.