Here, we identified caspase-2, protein kinase C (PKC)δ, and c-Jun NH₂-terminal kinase (JNK) as key components of the doxorubicin-induced apoptotic cascade. Using cells stably transfected with an antisense construct for caspase-2 (AS2) as well as a chemical caspase-2 inhibitor, we demonstrate that caspase-2 is required in doxorubicin-induced apoptosis. We also identified PKCδ as a novel caspase-2 substrate. PKCδ was cleaved/activated in a caspase-2–dependent manner after doxorubicin treatment both in cells and in vitro. PKCδ is furthermore required for efficient doxorubicin-induced apoptosis because its chemical inhibition as well as adenoviral expression of a kinase dead (KD) mutant of PKCδ severely attenuated doxorubicin-induced apoptosis. Furthermore, PKCδ and JNK inhibition show that PKCδ lies upstream of JNK in doxorubicin-induced death. Jnk-deficient mouse embryo fibroblasts (MEFs) were highly resistant to doxorubicin compared with wild type (WT), as were WT Jurkat cells treated with SP600125, further supporting the importance of JNK in doxorubicin-induced apoptosis. Chemical inhibitors for PKCδ and JNK do not synergize and do not function in doxorubicin-treated AS2 cells. Caspase-2, PKCδ, and JNK were furthermore implicated in doxorubicin-induced apoptosis of primary acute lymphoblastic leukemia blasts. The data thus support a sequential model involving caspase-2, PKCδ, and JNK signaling in response to doxorubicin, leading to the activation of Bak and execution of apoptosis.