Abstract Nucleotide-binding and oligomerization domain (NOD)–like receptors NOD1 and NOD2 are cytosolic innate immune receptors that recognize microbial peptidoglycans. Although studies have addressed the role of NOD proteins in innate immune responses, little attention has been given to their impact on the developing adaptive immune system. We have assessed the roles of NOD1 and NOD2 deficiency on T cell development in mice. Our results demonstrate that NOD1 and NOD2 promote the positive selection/maturation of CD8 single-positive thymocytes in a thymocyte-intrinsic manner. TCR-mediated ERK phosphorylation is significantly reduced in the absence of NOD proteins, but receptor-interacting protein 2 is not involved in CD8 single-positive thymocyte selection or ERK signaling. Commensal bacteria–free animals have thymocyte maturation defects, and exogenous NOD ligands can enhance thymocyte maturation in culture. These results raise the intriguing possibility that abnormal lymphocyte responses observed in NOD-dependent inflammatory diseases are not driven solely by microbial signals in the gut, but may also involve intrinsic lymphocyte defects resulting from impaired CD8 T cell thymic development.