Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow caused primarily by failure of normal homeostatic mechanisms to prevent the expansion of postgerminal center plasma cells. We have examined the molecular mechanisms that promote the survival of MM cells and have identified a key role for myeloid cell factor–1 (Mcl-1), an antiapoptotic member of the Bcl-2 family. These experiments were initiated by the observation that MM cells were exquisitely sensitive to culture in the presence of actinomycin D: caspase activation occurred within 3 hours of treatment and cells were not protected by interleukin-6, the main MM cell growth and survival factor. Actinomycin D–induced apoptosis was blocked by proteasome inhibitors, suggesting that a labile protein was required for MM cell survival. Further analysis demonstrated that Mcl-1 was likely to be the labile factor governing MM cell survival. Mcl-1 protein levels decreased rapidly after culture in the presence of actinomycin D in concordance with effector caspase activation, but addition of proteasome inhibitors reversed the loss of Mcl-1 and maintained cell viability. The levels of other antiapoptotic proteins, including Bcl-2 and members of the inhibitors-of-apoptosis family, were unaffected by these interventions. Furthermore, Mcl-1 antisense oligonucleotides caused a rapid down-regulation of Mcl-1 protein levels and the coincident induction of apoptosis, whereas overexpression of Mcl-1 delayed actinomycin D–induced apoptosis with kinetics that correlated with expression levels of Mcl-1. These data indicate that Mcl-1 expression is required for the survival of MM cells and may represent an important target for future therapeutics.