Donor-reactive memory T cells (Tmem) can play an important role in mediating graft rejection after transplantation. Transplant recipients acquire donor-reactive Tmem not only through prior sensitization with alloantigens but also through previous exposure to environmental pathogens that are cross-reactive with allogeneic peptide-MHC complexes. Current dogma suggests that most, if not all, Tmem responses are independent of the requirement for CD28 and/or CD154/CD40-mediated costimulation to mount a recall response. However, heterogeneity among Tmem is increasingly being appreciated, and one important factor known to impact the function and phenotype of Ag-specific T cell responses is the amount/duration of Ag exposure. Importantly, the impact of Ag exposure on development of costimulation independence is currently unknown. In this study, we interrogated the effect of decreased Ag amount/duration during priming on the ability of donor-reactive Tmem to mediate costimulation blockade-resistant rejection during a recall response after transplantation in a murine model. Recipients possessing donor-reactive Tmem responses that were generated under conditions of reduced Ag exposure exhibited similar frequencies of Ag-specific T cells at day 30 postinfection, but, strikingly, failed to mediate costimulation blockade-resistant rejection after challenge with an OVA-expressing skin graft. Thus, these data demonstrate the amount/duration of Ag exposure is a critical factor in determining Tmem's relative requirement for costimulation during the recall response after transplantation.