The transcription factor NF-κB plays a central role in regulating inflammation and apoptosis, making it a compelling target for drug development. We identified a small molecule inhibitor (ML120B) that specifically inhibits IKKβ, an Ikappa-B kinase that regulates NF-κB. IKKβ and NF-κB are required in vivo for prevention of TNFα-mediated apoptosis. ML120B sensitized mouse bone marrow progenitors and granulocytes, but not mature B cells to TNFα killing in vitro, and induced apoptosis in vivo in the bone marrow and spleen within 6 hours of a single oral dose. In vivo inhibition of IKKβ with ML120B resulted in depletion of thymocytes and B cells in all stages of development in the bone marrow but did not deplete granulocytes. TNF receptor–deficient mouse thymocytes and B cells were resistant to ML120B-induced depletion in vivo. Surprisingly, surviving bone marrow granulocytes expressed TNFR1 and TNFR2 after dosing in vivo with ML120B. Our results show that inhibition of IKKβ with a small molecule in vivo leads to rapid TNF-dependent depletion of T and B cells. This observation has several implications for potential use of IKKβ inhibitors for the treatment of inflammatory disease and cancer.