Engraftment of allogeneic bone marrow (BM) has been shown to induce tolerance to organs genotypically matched with the BM donor. Immune reconstitution after BM transplantation therefore involves re-establishment of a T cell pool tolerant to antigens present on both donor and host tissues. However, how hematopoietic grafts exert their influence over the regenerating immune system is not completely understood. Prior studies suggest that education of the newly arising T cell pool involves distinct contributions from donor and host stromal elements. Specifically, negative selection is thought to be mediated primarily by donor BM-derived antigen-presenting cells, whereas positive selection is dictated by radio-resistant host-derived thymic stromal cells. In this report we studied the effect of highly purified allogeneic hematopoietic stem cells (HSCs) on organ transplantation tolerance induction and immune reconstitution. In contrast to engraftment of BM that results in near-complete donor T cell chimerism, HSC engraftment results in mixed T cell chimerism. Nonetheless we observed that HSC grafts induce tolerance to donor-matched neonatal heart grafts, and one way the HSC grafts alter host immune responses is via deletion of newly arising donor as well as radiation-resistant host T cells. Furthermore, using an in vivo assay of graft rejection to study positive selection we made the unexpected observation that T cells in chimeric mice rejected grafts only in the context of the donor MHC type. These latter findings conflict with the conventionally held view that radio-resistant host elements primarily dictate positive selection.bone marrow transplantation ͉ MHC restriction ͉ mice T ransplantation of allogeneic bone marrow (BM) is known to alter immune responses in recipients so that tolerance is established to tissues matched with the genotype of the BM donors (1-3). Thus, the process of regeneration of the hematopoietic system involves the re-establishment of parameters that identify self-from nonself-antigens. The way in which BM grafts affect these changes is not completely understood. However, because T cells control antigen-specific immune responses the pathways that lead to regeneration of the peripheral T cell pool are central to immune reconstitution. T cell development after BM transplantation (BMT) is thought to recapitulate normal T cell ontogeny, which begins with the migration of BM-derived hematopoietic stem cells (HSCs) or more differentiated progenitors to the thymus (4). Within the thymus, under the influence of a specialized stromal microenvironment, progenitor T cells expand, differentiate, and undergo the rigorous processes of positive and negative selection (5-8). Positive selection results in survival of T cells with antigen receptors that corecognize self-MHC molecules plus foreign peptides. T cells whose receptors do not detect self-MHC molecules die, presumably by failure to receive critical differentiating signals. Negative selection involves the removal of potentially autoreactive T cells tha...