Since the beginning of vascularized solid organ transplantation in the last century, the goal after successful surgery has been to prevent the new organ from being rejected by the recipient’s immune system. To prevent transplant rejection, immunosuppressive drugs have been used, albeit suboptimally and with many side effects. Transplantation tolerance, if achieved, would allow the recipient to accept the transplant long-term following a short-term treatment in the absence of any long-term immunosuppression and is a long-standing goal of the transplantation field. To date, transplantation tolerance has been prospectively induced in a limited number of patients through regimens combining donor bone marrow and solid organ transplantation, and has also developed spontaneously in rare individuals initially treated with conventional immunosuppression. To broaden the applicability of tolerance to all patients, a better understanding of tolerance mechanisms is required. In this thesis, we investigated a mouse model of costimulation-blockade-induced tolerance to understand which mechanisms of tolerance were actively engaged to maintain and define robust tolerance. Tolerance exists on a gradient rather than as an all-or-none state—multiple mechanisms need to be simultaneously engaged in order to maintain robust tolerance. A loss of tolerance can occur if enough mechanisms are simultaneously disrupted, but as these re-engage, tolerance can spontaneously reemerge. Two additional elements of T cell tolerance have been newly identified to occur in this model. Together, these data broaden our understanding of tolerance and afford new avenues of research to investigate with the hope of ultimately achieving transplantation tolerance in the clinic.