L ymphocyte activation gene-3 (LAG-3) is a cell-surface molecule with diverse biologic effects on T cell function. We recently showed that LAG-3 signaling is important in CD4⁺ regulatory T cell suppression of autoimmune responses. Here, we demonstrate that LAG-3 maintains tolerance to self and tumor antigens via direct effects on CD8⁺ T cells using 2 murine systems. Naive CD8⁺ T cells express low levels of LAG-3, and expression increases upon antigen stimulation. Our data show increased levels of LAG-3 protein on antigen-specific CD8⁺ T cells within antigen-expressing organs or tumors. In vivo antibody blockade of LAG-3 or genetic ablation of the Lag-3 gene resulted in increased accumulation and effector function of antigen-specific CD8⁺ T cells within organs and tumors that express their cognate antigen. Most notably, combining LAG-3 blockade with specific antitumor vaccination resulted in a significant increase in activated CD8⁺ T cells in the tumor and disruption of the tumor parenchyma. A major component of this effect was CD4 independent and required LAG-3 expression by CD8⁺ T cells. Taken together, these data demonstrate a direct role for LAG-3 on CD8⁺ T cells and suggest that LAG-3 blockade may be a potential cancer treatment.