Apart from clonal deletion of self-reactive T cells in the thymus, Tregs are the major regulators of immune responses in the periphery and maintain a state of self-tolerance free from autoimmune diseases. Due to their inherent suppressive function, Tregs are being explored for their therapeutic potential in preventing autoimmunity and improving survival of allografts. This review provides recent updates on Treg biology and their use in animal as well as clinical models of transplantation. We discuss potential problems that limit the widespread clinical application of Tregs and provide future perspectives on how to optimize their medical use. Special consideration is given to methods by which Tregs should be isolated and expanded in order to facilitate clinical therapies. We also focus on recent discussions of Treg stability and plasticity, with specific insights into preventing the loss of Treg suppression by allotransplant-mediated inflammation.