A subpopulation of T cells harbors a suppressor phenotype and can significantly dampen autoreactive CD4+ and CD8+ T-cell responses. These regulatory T (Treg) cells, which can arise naturally in the thymus and encompass a CD25+CD4+ T-cell repertoire or be antigenically induced, are central players in the maintenance of self-tolerance. A plethora of O-cell costimulatory and accessory receptor molecules expressed by Treg and/or non-regulatory T cells, such as GITR, OX40, and CTLA-4, are involved in modulating the pathogenesis of numerous autoimmune disorders, transplant rejection, and tumor immunity, as well as the control of infections. Exciting new evidence shows that O-cell costimulators, some of which are identified as hopeful discriminative Treg-cell markers, appear to mediate Treg-cell homeostasis and function. Understanding the biological significance of the O-cell costimulatory molecules and the accessory molecules expressed by Treg cells is a prerequisite to better characterizing this regulatory T-cell population. We provide a synopsis of the current understanding of several costimulatory molecules that can orchestrate the function of both naturally arising and antigen-inducible Treg cells.