Inhibition of the transcription factor nuclear factor (NF)-κB activity leads to a reduction in numbers of CD8⁺ single-positive (SP) thymocytes, suggesting a selective role for NF-κB in these cells. To further explore the role of NF-κB in SP thymocytes, we utilized transgenic models that allowed either inhibition or activation of NF-κB. We showed that activation of NF-κB played an important role in the selection of major histocompatibility complex (MHC) class I-restricted CD8⁺ T cells. Surprisingly, NF-κB was not activated in positively selected CD4⁺ thymocytes, and inhibition of NF-κB did not perturb positive or negative selection of CD4⁺ cells. However, enforced activation of NF-κB via a constitutively active inhibitor of κB (IκB) kinase transgene led to a nearly complete deletion of CD4 cells by pushing positively selecting CD4⁺ cells into negative selection. These studies therefore revealed a surprising difference of NF-κB activation in CD4⁺ and CD8⁺ thymocytes and suggested that NF-κB contributes to the establishment of thresholds of signaling that determine positive or negative selection of thymocytes.