Invariant natural killer T (iNKT) cells are composed of at least three functionally distinct subsets, NKT1, NKT2 and NKT17. Through selective activation of these three iNKT effector subsets, iNKT cells can modulate immune responses and tissue homeostasis in different fashions. However, the developmental steps that drive iNKT cells into functional distinct subsets have not been elucidated, and thus their potential to be utilized in anti-cancer or autoimmune immunotherapies has not been realized, despite the fact that iNKT stimulatory lipids are well-tolerated in human trials. My dissertation research aims to fill this knowledge gap by investigating the following aspects of iNKT biology: 1) characterizing the multipotent progenitor for the iNKT effector subsets (in chapter 2); 2) isolating the critical factors that determine how individual iNKT subsets are derived, with a focus on NKT2 cells (in chapter 3); 3) characterizing how distinct iNKT effector subsets specifically modulate protective host immune responses (in chapter 2 & 3); and 4) technical improvement in advancing more accurate analysis of ex vivo iNKT cells (in chapter 4). Firstly, in chapter 2, I demonstrate that the small proportion of thymic iNKT cells that express CCR7 represent a multi-potent progenitor pool that gives rise to effector subsets within the thymus. These CCR7+ iNKT cells also emigrate from the thymus in a Klf2 dependent manner, undergo further maturation after reaching the periphery. Furthermore, Ccr7 deficiency impaired differentiation of iNKT effector subsets and localization to the medulla. Parabiosis and intra-thymic transfer showed that thymic NKT1 and NKT17 were resident-they were not derived from and did not contribute to the peripheral pool. Finally, each thymic iNKT effector subset produces distinct factors that influence T cell development. Secondly, previous studies showed IL-4 is produced by NKT2 cells in the thymus, where it conditions CD8+ T cells to become “memory like” amongst other effects in the steady state. However, the signals that cause NKT2 cells to constitutively produce IL-4 remain poorly defined, where in the chapter 3, these signals were investigated. Using histocytometry, IL-4 producing NKT2 cells were localized to the thymic medulla, suggesting medullary signals might instruct NKT2 cells to produce IL-4. Moreover, NKT2 cells receive and require TCR stimulation for continuous IL-4 production at steady state, since NKT2 cells lost IL-4 production when intra-thymically transferred into Cd1d deficient recipients. In bone marrow chimeric recipients, only hematopoietic, but not stromal APC, provided such stimulation. Furthermore, using different Cre-recombinase transgenic mouse strains to specifically target CD1d deficiency to various APC, together with the use of diphtheria toxin receptor (DTR) transgenic mouse strains to deplete various APC, we found that macrophages were the predominant cell to stimulate NKT2 IL-4 production. Lastly, it has been recently shown that high extracellular ATP concentrations or NAD-mediated P2RX7 ribosylation by the enzyme ARTC2.2 can induce P2RX7 pore formation and cell death. Because both ATP and NAD are released during tissue preparation for analysis, cell death through these pathways may compromise the analysis of iNKT. The expression of ARTC2.2 and P2RX7 on distinct iNKT subsets is unclear, however, as is the impact of recovery from other nonlymphoid sites. Therefore, in the chapter 4, I showed NKT1 cells express high levels of both ARTC2.2 and P2RX7 compared with NKT2, NKT17 cells. Furthermore, I demonstrated that ARTC2.2 blockade enhanced NKT1 recovery from nonlymphoid tissues during cell preparation. Moreover, blockade of this pathway was essential to preserve functionality, viability, and proliferation of iNKT cells. Therefore, short-term in vivo blockade of the ARTC2.2/P2RX7 axis permits much improved flow cytometry–based phenotyping and enumeration of murine iNKT from nonlymphoid tissues, and it represents a crucial step for functional studies of this population. Altogether, I believe the findings here provide a clearer understanding of how the lipid specific iNKT cells become effector subsets as well as a technical improvement for accurate analysis of these cells.