Terminally differentiated keratinocytes are dead enucleated squams. We showed previously that the mitochondria-dependent cell death pathway might be gradually activated as differentiation progresses. In this study, we demonstrated that protoporphyrin IX, staurosporine, and rotenone induced apoptotic-like changes in the mitochondria, and early differentiation of keratinocytes without inducing apoptosis. Kinetics studies established that differentiation-related changes, including growth arrest, flattened morphology, stratification, and keratin 10 (K10) expression, were downstream of mitochondrial depolarization and proliferation, reactive oxygen species (ROS) production, and release of cytochrome c and apoptosis-inducing factor. When these changes were prevented by overexpressing Bcl-2 or pharmacologically decreasing the ROS level, K10 upregulation was inhibited, implying that the differentiated phenotype and K10 expression require apoptotic mitochondria, ROS being the most likely differentiation-mediating factor. Our data also suggest that the same mitochondria-affecting stimuli can induce either differentiation or apoptosis, depending on the keratinocyte's competency to undergo differentiation, a competency that may be controlled by Bcl-2.