Abstract Vα14 invariant (Vα14i) NKT cells are a subset of regulatory T cells that utilize a semi-invariant TCR to recognize glycolipids associated with monomorphic CD1d molecules. During development in the thymus, CD4+CD8+ Vα14i NKT precursors recognizing endogenous CD1d-associated glycolipids on other CD4+CD8+ thymocytes are selected to undergo a maturation program involving sequential expression of CD44 and NK-related markers such as NK1.1. The molecular requirements for Vα14i NKT cell maturation, particularly at early developmental stages, remain poorly understood. In this study, we show that CD4-Cre-mediated T cell-specific inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biological activities, selectively impairs Vα14i NKT cell development without perturbing the development of conventional T cells. In the absence of c-Myc, Vα14i NKT cell precursors are blocked at an immature CD44lowNK1.1− stage in a cell autonomous fashion. Residual c-Myc-deficient immature Vα14i NKT cells appear to proliferate normally, cannot be rescued by transgenic expression of BCL-2, and exhibit characteristic features of immature Vα14i NKT cells such as high levels of preformed IL-4 mRNA and the transcription factor promyelocytic leukemia zinc finger. Collectively our data identify c-Myc as a critical transcription factor that selectively acts early in Vα14i NKT cell development to promote progression beyond the CD44lowNK1.1− precursor stage.