Regulatory T cells (Tregs) are long-lived cells that suppress immune responses in vivo in a dominant and antigen-specific manner. Therefore, therapeutic application of Tregs to control unwanted immune responses is an active area of investigation. Tregs can confer long-term protection against auto-inflammatory diseases in mouse models. They have also been shown to be effective in suppressing alloimmunity in models of graft-versus-host disease and organ transplantation. Building on extensive research in Treg biology and preclinical testing of therapeutic efficacy over the past decade, we are now at the point of evaluating the safety and efficacy of Treg therapy in humans. This review focuses on developing therapy for transplantation using CD4(+)Foxp3(+) Tregs, with an emphasis on the studies that have informed clinical approaches that aim to maximize the benefits while overcoming the challenges and risks of Treg cell therapy.