Vα14i natural killer T (NKT)–cell function has been implicated in a number of disease conditions. The molecular events that drive Vα14i NKT-cell development remain elusive. We recently showed that T-bet is required for the terminal maturation of these cells. Here we identify some of the genetic targets of T-bet during Vα14i NKT-cell lineage development. Microarray gene-expression analyses on developing Vα14i NKT cells were performed and provide a molecular framework to study these maturation events. In vitro ectopic expression of T-bet in immature Vα14i NKT cells, which do not yet express T-bet, was sufficient to promote Vα14i NKT-cell maturation, driving the expression of multiple genes, including those that participate in migration, survival, and effector functions. By regulating the expression of T-helper 1 (Th1)–associated cytokines, chemokines, chemokine receptors, and molecules involved in cytolysis, T-bet defines the unique lineage attributes of mature Vα14i NKT cells and acts to link these attributes to a developmental process.