The induction and maintenance of allograft tolerance is a daunting challenge. Although combined blockade of CD28 and CD40 ligand (CD40L)-costimulatory pathways prevents allograft rejection in some murine models, this strategy is unable to sustain engraftment in the most immunogenic allograft and strain combinations. By targeting T cell activation signals 1 and 2 with the novel combination of anti-CD45RB and anti-CD40L, we now demonstrate potent enhancement of engraftment in C57BL/6 recipients that are relatively resistant to costimulatory blockade. This combination significantly augments the induction of tolerance to islet allografts and dramatically prolongs primary skin allograft survival. Compared with either agent alone, anti-CD45RB plus anti-CD40L inhibits periislet infiltration by CD8 cells, B cells, and monocytes; inhibits Th1 cytokines; and increases Th2 cytokine expression within the graft. These data indicate that interference with activation signals one and two may provide synergy essential for prolonged engraftment in situations where costimulatory blockade is only partially effective.