Graft rejection by the immune system is a major cause of transplant failure. Lifelong immunosuppression decreases the incidence of graft rejection; however, nonspecific immunosuppression results in increased susceptibly to infection and cancer. Regulatory T cells (T_regs), which suppress the activation of the immune system and induce tolerance, are currently under evaluation for use in clinical transplantation. Ex vivo expanded polyclonal T_regs that are introduced into transplant recipients alter the balance of T effector cells to T_regs; however, experimental data suggest that alloantigen-specific T_regs would be more effective at preventing graft rejection. We have developed a method to enrich alloantigen-specific human T_regs based on the coexpression of activation markers, CD69 and CD71. These T_regs could be readily expanded in vitro and demonstrated potent antigen-specific suppression. In a humanized mouse model of alloimmune-mediated injury of human skin grafts, alloantigen-specific T_regs resulted in a significant reduction in clinically relevant indicators of dermal tissue injury when compared with polyclonal T_regs, restoring a histology comparable to healthy skin. This method of human allospecific T_reg selection should be scalable to the clinic. The improved in vivo efficacy of alloantigen-specific T_regs over polyclonal T_regs shown here suggests that generating “customized” T_regs with defined anti-donor allospecificities may improve current practice in clinical immunotherapy.