Xenotransplantation could overcome the severe shortage of allogeneic organs, a major factor limiting organ transplantation. Unfortunately, transplantation of organs from pigs, the most suitable potential donor species, results in hyperacute rejection in primate recipients, due to the presence of anti–Galα1-3Gal (Gal) natural antibodies (NAbs) in their sera. We evaluated the ability to tolerize anti-Gal NAb–producing B cells in α1,3-galactosyltransferase knockout (GalT KO) mice using bone marrow transplantation (BMT) from GalT^+/+ wild-type (WT) mice. Lasting mixed chimerism was achieved in KO mice by cotransplantation of GalT KO and WT marrow after lethal irradiation. The levels of anti-Gal NAb in sera of mixed chimeras were reduced markedly 2 wk after BMT, and became undetectable at later time points. Immunization with Gal^+/+ xenogeneic cells failed to stimulate anti-Gal antibody production in mixed chimeras, whereas the production of non–Gal-specific antixenoantigen antibodies was stimulated. An absence of anti-Gal–producing B cells was demonstrated by enzyme-linked immunospot assays in mixed KO+WT→ KO chimeras. Thus, mixed chimerism efficiently induces anti-Gal–specific B cell tolerance in addition to T cell tolerance, providing a single approach to overcoming both the humoral and the cellular immune barriers to discordant xenotransplantation.