AbstractNF-κB transcription factors are known to regulate the expression of a number of genes involved in T cell activation and function. Some evidence has suggested that they also play a role in T cell development. However, the role of NF-κB in Ag-induced thymocyte differentiation has not been directly addressed to date. Here we critically examine this role by employing DPK, a CD4+CD8+ thymocyte line that undergoes differentiation upon TCR engagement in a process that closely mimics positive selection. Expression of a degradation-resistant form of IκBα in DPK cells results in constitutive inhibition of NF-κB activity. We find that in the absence of NF-κB activity, MHC-peptide-induced differentiation of DPK is blocked. Furthermore, differentiation induced by a nonphysiologic stimulus, anti-TCR Ab, is greatly reduced. Altogether, our data indicate a requirement for NF-κB in the developmental changes associated with positive selection.