NKT cells are glycolipid-reactive lymphocytes that express markers and perform functions common to both T lymphocytes and NK cells. Although the genetic events controlling conventional T cell development are well defined, the transcription factors and genetic programs regulating NKT cell development are only beginning to be elucidated. Previously, we described the NKT cell-deficient phenotype of transgenic (Tg) mice constitutively expressing B cell-activating transcription factor (BATF), a basic leucine zipper protein and inhibitor of AP-1. In this study, we show that Tg BATF targets the majority of Valpha14Jalpha281 (Valpha14i(7)) NKT cells, regardless of CD4 expression and Vbeta gene usage. The residual NKT cells in the thymus of BATF-Tg mice are CD44(+), yet are slow to display the NK1.1 marker characteristic of mature cells. As a population, BATF-expressing NKT cells are TCRbeta/CD3epsilon(low), but express normal levels of CD69, suggesting a failure to expand appropriately following selection. Consistent with the sensitivity of NKT cells to BATF-induced changes in AP-1 activity, we detect a full complement of AP-1 basic leucine zipper proteins in wild-type NKT cells isolated from the thymus, spleen, and liver, and show that AP-1 DNA-binding activity and cytokine gene transcription are induced in NKT cells within a few hours of glycolipid Ag exposure. This study is the first to characterize AP-1 activity in NKT cells and implicates the integrity of this transcription factor complex in developmental events essential to the establishment of this unique T cell subset in the thymus.