AbstractThe cylindromatosis tumor suppressor gene (Cyld) encodes an enzyme (CYLD) with deubiquitinating activity that has been implicated in the regulation of thymocyte selection in an NF‐κB‐essential‐modulator (NEMO)‐dependent manner. The main known molecular defects in thymocytes with inactive CYLD (LckCre‐Cyldflx9/flx9) are the aberrant hyperactivation of NF‐κB and JNK pathways. In order to dissect further the molecular mechanism of CYLD‐dependent thymocyte selection and address the role of NF‐κB specifically, we generated double mutant mice (LckCre‐Cyldflx9/flx9‐Ikk2flx/flx) in which CYLD was inactivated concomitantly with IKK2 (IκB‐kinase 2) in thymocytes. Interestingly, thymic development and NF‐κB activity in double mutant mice were fully restored, indicating that an IKK2‐dependent function of CYLD that leads to the hyperactivation of the NF‐κB pathway is primarily responsible for the defective selection of thymocytes. Intriguingly, we observed a greater reduction of CD4+ and CD8+ T cells in the periphery of LckCre‐Cyldflx9/flx9‐Ikk2flx/flx mice compared with LckCre‐Ikk2flx/flx mice. Collectively, our data establish CYLD as a critical regulator of thymocyte selection in a manner that depends on IKK2 and NF‐κB activation. In addition, our data uncover an IKK2‐independent role for CYLD in the establishment of physiological T‐cell populations in the periphery.