Autoimmune diseases are debilitating conditions that pose a significant burden worldwide. T-cells are thought to play important roles in the coordination and development of immune responses in both health and disease. A key checkpoint in the prevention of inappropriate activation of T-cells is the requirement for co-stimulation by professional APCs via receptors such as CD28. The requirement for CD28 engagement for complete activation of T-cells is lost in Cbl-b mutants, which also develop multi-organ autoimmunity and are highly-susceptible to experimental autoimmune conditions, suggesting Cbl-b may therefore play a role in the generation or maintenance of peripheral T-cell tolerance. By subjecting Cbl-b mutant mice to well-characterized in vivo tolerization protocols, we find that T-cells from these mice, unlike those from wild type mice, maintain and intensify subsequent responsiveness both in vivo and in vitro, resulting in lethality. Thus, Cbl-b is indeed essential for the induction of immunotolerance to specific antigens.