Blockade of the CD40-CD154 costimulatory pathway can inhibit CD4+ T cell-mediated alloimmune responses. The aim of this study was to define the in vivo requirement for CD40-CD154 costimulation by CD4+ T cells that respond to alloantigen following direct recognition. We used TCR-transgenic CD4+ T cells that are reactive to the MHC class II alloantigen, H2A s. An experimental in vivo model was established that allowed direct comparison of the fate of a trace population of H2A s-reactive CD4+ T cells when challenged with different forms of H2A s+ alloantigen under conditions of CD40-CD154 costimulation blockade. In this study, we demonstrate that an iv infusion of H2A s+ leukocytes in combination with anti-CD154 therapy rapidly deletes H2A s-reactive CD4+ T cells. In contrast, following transplantation of an H2A s+ cardiac allograft, H2A s-reactive CD4+ T cell responses were unaffected by blocking CD40-CD154 interactions. Consistent with these findings, combined treatment with donor leukocytes and anti-CD154 therapy was found to be more effective in prolonging the survival of cardiac allografts compared with CD154 mAb treatment alone. The dominant mechanism by which donor leukocyte infusion and anti-CD154 therapy facilitate allograft acceptance is deletion of donor-reactive direct pathway T cells. No evidence for the generation of regulatory cells by this combined therapy was found. Taken together, these results clearly demonstrate that naive alloreactive CD4+ T cells have distinct requirements for CD40-CD154 costimulation depending on the form and microenvironment of primary alloantigen contact.