Reactive oxygen species (ROS) influence diverse cellular processes, including proliferation and apoptosis. Both endogenous and exogenous ROS activate signaling through mitogen-activated proteins kinase (MAPK) pathways, including those involving extracellular signal-regulated kinases (ERKs) or c-Jun N-terminal kinases (JNKs). Whereas low concentrations of ROS generally stimulate proliferation, high concentrations result in cell death. We found that low concentrations of ROS induced activating phosphorylation of ERKs, whereas high concentrations of ROS induced activating phosphorylation of JNKs. Mixed lineage kinase 3 (MLK3, also known as MAP3K11) directly phosphorylates JNKs and may control activation of ERKs. Mathematical modeling of MAPK networks revealed a positive feedback loop involving MLK3 that determined the relative phosphorylation of ERKs and JNKs by ROS. Cells exposed to an MLK3 inhibitor or cells in which MLK3 was knocked down showed increased activation of ERKs and decreased activation of JNKs and were resistant to cell death when exposed to high concentrations of ROS. Thus, the data indicated that MLK3 is a critical factor controlling the activity of kinase networks that control the cellular responses to different concentrations of ROS.