The nuclear factor (NF)-I?B signalling pathway is known to be critical for natural killer T (NKT) cell differentiation; however, the role of individual NF-I?B transcription factors and the precise developmental stages that they control remain unclear. We have investigated the influence of the classical NF-I?B transcription factors NF-I?B1, c-Rel and RelA on NKT cell development and function, using gene-deleted mice. Individually, none of these factors were essential for the requirement of NF-I?B signalling in early NKT cell development before NK1.1 expression, in contrast to earlier reports in which the classical NF-I?B pathway was globally disrupted. Instead, we found that each factor played a non-redundant role in later stages of NKT cell maturation and function. Although NF-I?B1 deficiency resulted in a moderate reduction in mature NK1.1+ NKT cells, this was found to be more subtle than previously reported. RelA deficiency had a more profound effect on the NK1.1+ stage of NKT cell development, whereas c-Rel-deficient mice had normal NKT cell numbers. All three factors (NF-I?B1, RelA and c-Rel) were necessary for normal NKT cell cytokine production. Notably, IL-17, which is produced by a specific subset of NKT cells (NKT-17 cells), defined as NK1.1 -CD4-, was not impaired by a lack of these individual NF-I?B transcription factors, nor was this subset depleted, suggesting that NKT-17 cells are regulated independently of the NF-I?B pathway. Thus, individual NF-I?B family members have a largely redundant role in early NKT cell development, but each of them has an important and distinct role in NKT cell maturation and/or function. A? 2011 Australasian Society for Immunology Inc. All rights reserved.