Abstract Transcription factor NF-κB controls the expression of multiple genes involved in immunity and inflammation. The initial activation and duration of NF-κB signaling is regulated by posttranslational modifications to IκB kinase, which earmarks inhibitors of NF-κB for degradation. Prior studies suggest that K63-linked ubiquitination of NEMO (NF-κB essential modulator), an IκB kinase regulatory subunit, is critical for NF-κB and MAPK signaling following engagement of Ag receptors. We now demonstrate that NF-κB and MAPK pathways are largely unaffected in primary cells from mice harboring a ubiquitination-defective form of NEMO, NEMO-KR. TLR- but not Ag receptor-induced cellular responses are impaired in NEMO-KR mice, which are more resistant to LPS-induced endotoxic shock than wild-type animals. Thus, one function of NEMO ubiquitination is to fine tune innate immune responses under TLR control.