The S1P₁ receptor, on the surface of lymphocytes and endothelial cells, regulates the unique trafficking behavior of certain lymphocyte populations. We have examined whether the S1P₁ receptor also dictates the distinctive tissue distribution of V14-J18 natural killer T (NKT) cells, whose trafficking pattern is not well understood. Mice (TCS1P₁KO) were established with a conditional deletion of the S1P₁ receptor in thymocytes that included precursors of NKT cells. Within the thymus, NKT cells were found at normal or increased levels, indicating that S1P₁ receptor expression was dispensable for NKT cell development. However, substantially reduced numbers of NKT cells were detected in the peripheral tissues of the TCS1P₁KO mice. Short-term S1P₁ deletion after NKT cells had established residence in the periphery did not substantially alter their distribution in tissues, except for a partial decrease in the spleen. FTY720, a S1P₁ receptor ligand that has potent effects on the trafficking of conventional T cells, did not alter the preexisting distribution of NKT cells within peripheral tissues of wild-type mice. Our results indicate that the S1P₁ receptor expression on NKT cells is dispensable for development within thymus but is essential for the establishment of their tissue residency in the periphery.—Allende, M. L., Zhou, D., Kalkofen, D. N., Benhamed, S., Tuymetova, G., Borowski, C., Bendelac, A., Proia, R. L. S1P₁ receptor expression regulates emergence of NKT cells in peripheral tissues.