Costimulatory blockade can induce long-term allograft survival in naïve animals, but may not be as effective in animals with previously primed immune repertoires. We attempted to induce long-term graft survival in B10.D2 recipients of B10.A cardiac allografts using donor-specific transfusion (DST) plus anti-CD40 ligand antibody (αCD40L). Recipients were either naïve mice, or mice previously primed to B10.A or third party alloantigens through engraftment and rejection of skin transplants. Untreated naïve mice rejected cardiac transplants by day 15 and contained a high frequency of primed, donor-reactive T cells. Donor-specific transfusion/αCD40L treatment of naïve animals induced long-term graft survival associated with low frequencies of donor-reactive T cells. Previous priming of donor-specific T cells through rejection of B10.A, but not third party, skin grafts prevented the effects of DST/αCD40L on prolonging survival of B10.A hearts. Moreover, adoptive transfer of CD3+, CD4+ or CD8+ T cells from B10.A skin-graft-primed animals prevented the effects of DST/αCD40L. The data demonstrate that animals with immune repertoires containing previously primed, donor-reactive T cells are resistant to the effects of costimulatory blockade. The findings have important implications for ongoing, costimulatory blockade-based trials in humans, whose T-cell repertoires are known to contain memory alloreactive T cells.