After a brief period of antigenic stimulation, T cells become committed to a program of autonomous expansion and differentiation. We investigated the role of antigen-specific T cell precursor frequency as a possible cell-extrinsic factor impacting T cell programming in a model of allogeneic tissue transplantation. Using an adoptive transfer system to incrementally raise the precursor frequency of antigen-specific CD8⁺ T cells, we found that donor-reactive T cells primed at low frequency exhibited increased cellular division, decreased development of multifunctional effector activity, and an increased requirement for CD28- and CD154-mediated costimulation relative to those primed at high frequency. The results demonstrated that recipients with low CD4⁺ and CD8⁺ donor-reactive T cell frequencies exhibited long-term skin graft survival upon CD28/CD154 blockade, whereas simultaneously raising the frequency of CD4⁺ T cells to ∼0.5% and CD8⁺ T cells to ∼5% precipitated graft rejection despite CD28/CD154 blockade. Antigenic rechallenge of equal numbers of cells stimulated at high or low frequency revealed that cells retained an imprint of the frequency at which they were primed. These results demonstrate a critical role for initial precursor frequency in determining the CD8⁺ T cell requirement for CD28- and CD154-mediated costimulatory signals during graft rejection.