Invariant Vα14i NKT (iNKT) cells are a specialized subset of T lymphocytes with regulatory functions. They coexpress TCRαβ and natural killer cell markers. They differentiate through interaction of their Vα14-Jα18 invariant TCRα chains with CD1d expressed on double-positive (DP) thymocytes. Although their development has been shown to be thymus dependent, their developmental pathway has not been definitively established. By using genetic analyses, we show here that all iNKT cells are selected from a pool of DP thymocytes. Their development is absolutely dependent on Runx1 and RORγt, transcription factors that influence, but are not required for, development of conventional T cells. Our results indicate that even though CD1d binding DP thymocytes have yet to be observed, Vα14-Jα18 rearrangement in these cells is required for development of iNKT cells.