The regulatory role of the thymic microenvironment during trafficking and differentiation of the invariant NKT (iNKT) cell lineage remains poorly understood. In this study, we show that fractalkine receptor expression marks emigrating subpopulations of the NKT1, NKT2, and NKT17 sublineages in the thymus and peripheral organs of naive mice. Moreover, NKT1 sublineage cells can be subdivided into two subsets, namely NKT1(a) and NKT1(b), which exhibit distinct developmental and tissue-specific distribution profiles. More specifically, development and trafficking of the NKT1(a) subset are selectively dependent upon lymphotoxin (LT)alpha 1 beta 2-LT beta receptor-dependent differentiation of thymic stroma, whereas the NKT1(b), NKT2, and NKT17 sublineages are not. Furthermore, we identify a potential cellular source for LT alpha 1 beta 2 during thymic organogenesis, marked by expression of IL-7R alpha, which promotes differentiation of the NKT1(a) subset in a noncell-autonomousmanner. Collectively, we propose a mechanism by which thymic differentiation and retention of the NKT1 sublineage are developmentally coupled to LT alpha 1 beta 2-LT beta receptor-dependent thymic organogenesis.