Cells lacking functional NF-kappaB die after ligation of some tumor necrosis factor (TNF) receptor family members through failure to express NF-kappaB-dependent anti-apoptotic genes. NF-kappaB activation requires the IkappaB kinase (IKK) complex containing two catalytic subunits named IKKalpha and IKKbeta that regulate distinct NF-kappaB pathways. IKKbeta is critical for classical signaling that induces pro-inflammatory and anti-apoptotic gene profiles, whereas IKKalpha regulates the non-canonical pathway involved in lymphoid organogenesis and B-cell development. To determine whether IKKalpha and IKKbeta differentially function in rescuing cells from death induced by activators of the classical and non-canonical pathways, we analyzed death after ligation of the TNF and lymphotoxin-beta receptors, respectively. Using murine embryonic fibroblasts (MEFs) lacking each of the IKKs, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, and dominant negative Fas-associated death domain protein, we found that deletion of these kinases sensitized MEFs to distinct cell death pathways. MEFs lacking IKKalpha were sensitized to death in response to both cytokines that was entirely caspase-dependent, demonstrating that IKKalpha functions in this process. Surprisingly, death of IKKbeta-/- MEFs was not blocked by caspase inhibition, demonstrating that IKKbeta negatively regulates caspase-independent cell death (CICD). CICD was strongly activated by both TNF and lymphotoxin-beta receptor ligation in IKKbeta-/- MEFs and was accompanied by loss of mitochondrial membrane potential and the generation of reactive oxygen species. CICD was inhibited by the anti-oxidant butylated hydroxyanosole and overexpression of Bcl-2, neither of which blocked caspase-dependent apoptosis. Our findings, therefore, demonstrate that both IKKalpha and IKKbeta regulate cytokine-induced apoptosis, and IKKbeta additionally represses reactive oxygen species- and mitochondrial-dependent CICD.