CD1d antigen presentation to natural killer T (NKT) cells expressing the semi-invariant T cell receptor V_α14J_α18 requires CD1d trafficking through endosomal compartments; however, the endosomal events remain undefined. We show that mice lacking the endosomal protease cathepsin L (catL) have greatly reduced numbers of V_α14⁺NK1.1⁺ T cells. In addition, catL expression in thymocytes is critical not only for selection of these cells in vivo but also for stimulation of V_α14⁺NK1.1⁺ T cells in vitro. CD1d cell-surface expression and intracellular localization appear normal in catL-deficient thymocytes, as does the lysosomal morphology; this implies a specific role for catL in regulating presentation of natural CD1d ligands mediating V_α14⁺NK1.1⁺ T cell selection. These data implicate lysosomal proteases as key regulators of not only classical major histocompatibility complex class II antigen presentation but also nonclassical CD1d presentation.