To prevent the occurrence and expansion of autoreactive T cell clones, and hence the development of autoimmune disease (such as multiple sclerosis, rheumatoid arthritis, lupus erythematosus, and diabetes), the healthy immune system is equipped with a variety of immunological mechanisms, summarized as self-tolerance. The strength and frequency of the T cell receptor (TCR) signal as well as the costimulatory context critically control T cell fate and the composition of the T cell repertoire at different stages of lymphoid development. Central tolerance mechanisms in the thymus cause developing T cells with high affinity for self-peptide-MHC to undergo clonal deletion via programmed cell death (apoptosis) in a process referred to as negative selection. In contrast, T cells with a weak affinity are positively selected or, if the signal is just below the deletional threshold, enter a pathway to become regulatory T cells (Treg). Peripheral tolerance involves functional inactivation (“anergy”), suppression or extrathymic clonal deletion of autoreactive T cells that have escaped negative selection. The activation thresholds of TCR- and costimulatory-signaling pathways are modified by different groups of negative regulatory proteins with distinct developmentally regulated expression/activation patterns and functional mechanisms. This review provides an overview of the key negative regulators of T cell receptor signaling, including the Cbl family of ubiquitin ligases, phosphatases, the Dok family of adaptor proteins, as well as the serinethreonine kinase Drak2, and compares their differential impact on central and peripheral tolerance mechanisms, including clonal deletion, anergy and Treg suppression. Furthermore, the impact of targeting the related genes in mice on the susceptibility to different autoimmune diseases as well as the therapeutic potential of developing specific and effective immune treatments that modify these negative regulators of TCR signaling are explored.