Reactive oxygen species (ROS), a byproduct of cellular metabolism, damage intracellular macromolecules and, when present in excess, can promote normal hematopoietic stem cell differentiation and exhaustion. However, mechanisms that regulate the amount of ROS in leukemia-initiating cells (LICs) and the biological role of ROS in these cells are largely unknown. We show here that the ROS(low) subset of CD44(+) cells in T cell acute lymphoblastic leukemia (T-ALL), a malignancy of immature T cell progenitors, is highly enriched in the most aggressive LICs and that ROS accumulation is restrained by downregulation of protein kinase C θ (PKC-θ). Notably, primary mouse T-ALLs lacking PKC-θ show improved LIC activity, whereas enforced PKC-θ expression in both mouse and human primary T-ALLs compromised LIC activity. We also show that PKC-θ is regulated by a new pathway in which NOTCH1 induces runt-related transcription factor 3 (RUNX3), RUNX3 represses RUNX1 and RUNX1 induces PKC-θ. NOTCH1, which is frequently activated by mutation in T-ALL and required for LIC activity in both mouse and human models, thus acts to repress PKC-θ. These results reveal key functional roles for PKC-θ and ROS in T-ALL and suggest that aggressive biological behavior in vivo could be limited by therapeutic strategies that promote PKC-θ expression or activity, or the accumulation of ROS.