Arsenic trioxide induces c-jun N-terminal kinase (JNK) activation and apoptosis in acute promyelocytic leukemia (APL), where it has major clinical activity, but whether JNK is necessary to induce apoptosis is unknown. To clarify this necessity, we established 2 arsenic trioxide (As₂O₃)-resistant subclones of the APL cell line, NB4. Both resistant lines showed little activation of JNK1 following treatment with As₂O₃, even at doses sufficient to elicit robust activation in NB4 cells. One mechanism of resistance in these cells is up-regulated glutathione (GSH) content, and GSH depletion by l-buthionine-[S,R]-sulfoximine (BSO) restores JNK activation and As₂O₃ sensitivity. This correlation between JNK activation and apoptosis led us to test whether inhibition of JNK would protect cells from As₂O₃-induced apoptosis. SEK1^-/- mouse embryo fibroblasts (MEFs) showed diminished JNK activation following As₂O₃ treatment and were protected from As₂O₃-induced but not doxorubicin-induced apoptosis. Furthermore, treatment of arsenic trioxide-sensitive APL cells with the JNK inhibitor, dicumarol, significantly increased growth and survival in response to As₂O₃ but did not protect cells from doxorubicin. Together, these data support an essential role for JNK signaling in the induction of growth inhibition and apoptosis by As₂O₃ and suggest that activating JNK may provide a therapeutic advantage in the treatment of cancers that do not respond to arsenic alone. (Blood. 2004;103:3496-3502)