Mature T cells must discriminate between brief interactions with self-peptides and prolonged binding to agonists. The kinetic proofreading model posits that certain T-cell antigen receptor signaling nodes serve as molecular timers to facilitate such discrimination. However, the physiological significance of this regulatory mechanism and the pathological consequences of disrupting it are unknown. Here we report that accelerating the normally slow phosphorylation of the linker for activation of T cells (LAT) residue Y136 by introducing an adjacent Gly135Asp alteration (LAT^G135D) disrupts ligand discrimination in vivo. The enhanced self-reactivity of LAT^G135D T cells triggers excessive thymic negative selection and promotes T-cell anergy. During Listeria infection, LAT^G135D T cells expand more than wild-type counterparts in response to very weak stimuli but display an imbalance between effector and memory responses. Moreover, despite their enhanced engagement of central and peripheral tolerance mechanisms, mice bearing LAT^G135D show features associated with autoimmunity and immunopathology. Our data reveal the importance of kinetic proofreading in balancing tolerance and immunity.