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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 Vα14‐Jα18 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 …