Invariant Natural Killer T (iNKT) cells are unconventional T cells that can recognize lipid antigens that are otherwise invisible to conventional T cells.1 In mice, these cells are restricted to the MHC Class Ib molecule, CD1d, and play key roles in a multitude of immune-mediated diseases.2 The Notch signaling pathway is indispensable for the lineage commitment and early differentiation of T cells.3 However, its specific role in the development, differentiation, and effector function of iNKT cells remains poorly understood.8,9 iNKT cells differentiate into three major effector subsets - iNKT1, 2 and 17, based on their transcription factor expression and cytokine production profiles.4 The spatiotemporal emergence of these different subsets also remains a relatively unexplored aspect of iNKT cell biology.5 Using a mouse model3,6,7 that allows precise temporal control over Notch responsiveness, and therefore T cell development, we study the role of Notch signaling on the development, cell subset differentiation, and effector function of mouse iNKT cells. This mouse model also enables us to analyze T cell development in real-time, allowing us to track the emergence of different cell subsets of iNKT cells during ontogeny. Using bone-marrow chimeras, we have also compared the development of these “Notch-inducible” iNKT cells with or without Notch responsiveness in an intact thymic environment. A deeper understanding of the iNKT cell differentiation process, and the role of Notch signaling in these processes, could have important implications in determining disease susceptibility and developing better systems to generate iNKT cells for therapeutic applications.