The crucial role of regulatory T (Treg) cells in self-tolerance and downregulating immune responses has been clearly established. Numerous different Treg subsets have been identified that possess distinct phenotypes and functions in various disease models. Among these subsets, alphabeta-TCR+CD3+CD4-CD8- double-negative (DN) Treg cells have been shown to be able to inhibit a variety of immune responses in part via direct killing of effector T cells in an antigenspecific manner in both mice and humans. This was shown to occur at least partially by acquisition of MHC-peptide complexes from antigen-presenting cells (APCs) and subsequent Fas/Fas-ligand interactions. In addition, DN Treg cells have been shown to express several molecules uncommon to other Treg cell subsets, such as IFN-gamma, TNF-alpha, Ly6A, FcRgamma, and CXCR5, which may contribute to their unique regulatory ability. Understanding the development and regulatory functions of DN Treg cells may elucidate the etiology for loss of self-tolerance and serve as a therapeutic modality for various diseases. This review will summarize the characteristics, developmental pathways, and mechanisms of action of DN Treg cells, as well as their role in transplant tolerance, autoimmunity, and anticancer immunity.