Induction of tolerance is likely to be essential for successful xenotransplantation because immune responses across xenogeneic barriers are vigorous. Although mixed hematopoietic chimerism leads to stable donor-specific tolerance in allogeneic and closely related xenogeneic (eg, rat-to-mouse) combinations, the ability of this approach to induce tolerance across a highly disparate xenogeneic barrier has not yet been demonstrated. In this study, we investigated the immune responses of murine T cells that developed in mice with pre-established porcine hematopoietic chimerism. Our results show for the first time that induction of porcine hematopoietic chimerism can eliminate the development of antiporcine donor responses in a highly disparate xenogeneic species. Porcine hematopoietic chimeras showed donor-specific nonresponsiveness in the mixed lymphocyte reaction, lack of antidonor IgG antibody production, and acceptance of donor skin grafts. Thus, mixed chimerism is capable of inducing tolerance in a highly disparate xenogeneic combination and may have clinical potential to prevent xenograft rejection.