Reactive oxygen species (ROS) can be generated following cell stimulation and function as intracellular signaling molecules. To determine signaling components involved in ROS induction, human U937 blood cells grown in 10% serum were exposed to serum-free media. It was previously reported that serum withdrawal (SW) killed cells by elevating cellular ROS levels. This study showed that SW activates phosphoinositide 3-kinase (PI3K). PI3K activation was evident after the ROS levels began increasing, and an antioxidant blockade of this increase resulted in PI3K activation suppression. Interestingly, the inhibition of PI3K activity/activation using either its specific inhibitor or dominant-negative mutant attenuated the subsequent additional increase in the ROS levels. These results suggest that SW-induced ROS activate PI3K, which in turn promotes the process leading to ROS accumulation. The present study also revealed that both ROS and PI3K support SW-induced cell death by activating stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). Overall, it appears that SW triggers a positive mutual interaction between ROS and PI3K, which amplifies signals required for the induction of an SAPK-dependent death pathway.