Trophic factor deprivation (TFD) activates c-Jun N-terminal kinases (JNKs), culminating in coordinate AP1-dependent transactivation of the BH3-only BCL-2 proteins BIM_EL and HRK, which in turn are critical for BAX-dependent cytochrome c release, caspase activation, and apoptosis. Here, we report that TFD caused not only induction but also phosphorylation of BIM_EL. Mitochondrially localized JNKs but not upstream activators, like mixed-lineage kinases (MLKs) or mitogen-activated protein kinase kinases (MKKs), specifically phosphorylated BIM_EL at Ser65, potentiating its proapoptotic activity. Inhibition of the JNK pathway attenuated BIM_EL expression, prevented BIM_EL phosphorylation, and abrogated TFD-induced apoptosis. Conversely, activation of this pathway promoted BIM_EL expression and phosphorylation, causing BIM- and BAX-dependent cell death. Thus, JNKs regulate the proapoptotic activity of BIM_EL during TFD, both transcriptionally and posttranslationally.