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G alα1,3Gal–reactive (Gal-reactive) antibodies are a major impediment to pig-to-human xenotransplantation. We investigated the potential to induce tolerance of anti-Gal–producing cells and prevent rejection of vascularized grafts in the combination of α1,3-galactosyltransferase wild-type (GalT^+/+) and deficient (GalT^–/–) mice. Allogeneic (H-2 mismatched) GalT^+/+ bone marrow transplantation (BMT) to GalT^–/– mice conditioned with a nonmyeloablative regimen, consisting of depleting CD4 and CD8 mAb’s and 3 Gy whole-body irradiation and 7 Gy thymic irradiation, led to lasting multilineage H-2^bxd GalT^+/+ + H-2^d GalT^–/– mixed chimerism. Induction of mixed chimerism was associated with a rapid reduction of serum anti-Gal naturally occurring antibody levels. Anti-Gal–producing cells were undetectable by 2 weeks after BMT, suggesting that anti-Gal–producing cells preexisting at the time of BMT are rapidly tolerized. Even after immunization with Gal-bearing xenogeneic cells, mixed chimeras were devoid of anti-Gal–producing cells and permanently accepted donor-type GalT^+/+ heart grafts (>150 days), whereas non-BMT control animals rejected these hearts within 1–7 days. B cells bearing receptors for Gal were completely absent from the spleens of mixed chimeras, suggesting that clonal deletion and/or receptor editing may maintain B-cell tolerance to Gal. These findings demonstrate the principle that induction of mixed hematopoietic chimerism with a potentially relevant nonmyeloablative regimen can simultaneously lead to tolerance among both T cells and Gal-reactive B cells, thus preventing vascularized xenograft rejection.