Although transplantation is the treatment of choice for end stage organ failure, transplanted organs are vulnerable to immune-mediated rejection. To prevent rejection, patients are maintained on a strict immunosuppressive regimen; however, immunosuppression is costly, and the side effects associated with continuing life-long immunosuppression are numerous and unpleasant, leading to patient noncompliance. Current research efforts aim to replace conventional immunosuppression with short-term treatments inducing donor-specific immune tolerance, which would mean a patient is only nonresponsive to transplant-derived antigen and would retain their ability to mount immune responses to other antigens, such as those from infections and malignancies. Although possible to achieve in patients, donor-specific tolerance is rare and remains vulnerable to inflammatory challenges capable of precipitating transplant rejection. In this dissertation, we investigated vulnerabilities to donor-specific transplantation tolerance during both the induction and maintenance phases of tolerance. We identified functional heterogeneity within the transplant-reactive T cell compartment, dependent upon the length of their cognate antigen exposure in the context of immunosuppression: persistent antigen promoted T cell dysfunction whereas T cells specific for non-persistent antigens retained function. By forcing antigen exposure, we promoted widespread T cell dysfunction and protected the transplant recipients from infection-mediated rejection. We also investigated the memory T cell response following treatment with persistent antigen and immunosuppression and identified that memory T cells can develop a degree of dysfunction, although not to the same extent as naïve T cells. As robust allospecific T cell dysfunction is a hallmark of tolerance and desirable in patients, these findings may inform future strategies to promote clinical transplantation tolerance.