Sensitization is a critical unresolved challenge in transplantation. We show for the first time that blockade of CD154 alone or combined with T-cell depletion prevents sensitization. Allogeneic skin grafts were rejected by recipients treated with anti-alphabeta T-cell receptor (TCR), anti-CD154, anti-OX40L, or anti-inducible costimulatory pathway (ICOS) mAb alone with a kinetic similar to untreated recipients. However, the production of anti-donor MHC antibody was prevented in mice treated with anti-CD154 mAb only, suggesting a specific role for the CD154-CD40 pathway in B-cell activation. The impairment of T cell-dependent B-cell responses by blocking CD154 occurs through inhibiting activation of T and B cells and secretion of IFN-gamma and IL-10. Combined treatment with both anti-CD154 and anti-alphabeta TCR abrogated antidonor antibody production and resulted in prolonged skin graft survival, suggesting the induction of both T- and B-cell tolerance with prevention of allogeneic sensitization. In addition, we show that the tolerance induced by combined treatment was nondeletional. Moreover, these sensitization-preventive strategies promote bone marrow engraftment in recipients previously exposed to donor alloantigen. These findings may be clinically relevant to prevent allosensitization with minimal toxicity and point to humoral immunity as playing a dominant role in alloreactivity in sensitized recipients.