The role of Bim in synergistic interactions between UCN-01 and MEK1/2 inhibitors in human multiple myeloma cells was investigated. Exposure of U266 or RPMI8226 cells to UCN-01 resulted in ERK1/2 activation-associated BimEL phosphorylation/downregulation, events abrogated by MEK1/2 inhibitors. Enforced activation of ERK1/2 by transfection with constitutively active-MEK1 diminished the capacity of PD98059 but not PD184352 to block UCN-01-mediated BimEL phosphorylation and to potentiate apoptosis. Co-treatment with MEK1/2 inhibitors increased the association of BimEL with both Bcl-2 and Bcl-xL in UCN-01-treated cells, leading to Bax/Bak conformational change and Bax mitochondrial translocation. Downregulation of BimEL by shRNA substantially diminished UCN-01/MEK inhibitor-mediated Bax/Bak activation and apoptosis. Furthermore, transfection of cells with S65A Bim, a mutant resistant to UCN-01-mediated phosphorylation, significantly sensitized cells to UCN-01 lethality. Conversely, ectopic expression of either Bcl-2 or Bcl-xL did not alter UCN-01/MEK1/2 inhibitor-mediated modifications in BimEL phosphorylation, but largely prevented cell death. Finally, IL-6 or IGF-1 failed to prevent MEK1/2 inhibitors from blocking UCN-01-induced BimEL phosphorylation/degradation or cell death. Collectively, these findings argue that UCN-01-mediated ERK1/2 activation leads to BimEL phosphorylation/inactivation, resulting in cytoprotection, and that interference with these events by MEK1/2 inhibitors plays a critical role in synergistic induction of apoptosis by these agents.