Murine CD1 has been implicated in the development and function of an unusual subset of T cells, termed natural T (NT) cells, that coexpress the T cell receptor (TCR) and the natural killer cell receptor NK1.1. Activated NT cells promptly produce large amounts of IL-4, suggesting that these cells can influence the differentiation of CD4⁺ effector T cell subsets. We have generated mice that carry a mutant CD1d1 gene. NT cell numbers in the thymus, spleen, and liver of these mice were dramatically reduced. Activated splenocytes from mutant mice did not produce IL-4, whereas similarly treated wild-type splenocytes secreted large amounts of this cytokine. These results demonstrate a critical role for CD1 in the positive selection and function of NT cells.