Reactive oxygen species (ROS), a by-product of cellular metabolism, damage intracellular macromolecules and, in excess, can promote normal hematopoietic stem cell differentiation and exhaustion 1 – 3 . However, mechanisms that regulate ROS levels in leukemia-initiating cells (LICs) and the biological role of ROS in these cells remain largely unknown. We show here the ROS low subset of CD44 + cells in T-cell acute lymphoblastic leukemia (T-ALL), a malignancy of immature T-cell progenitors, to be highly enriched in the most aggressive LICs, and that ROS are maintained at low levels by downregulation of protein kinase C theta (PKCθ). Strikingly, primary mouse T-ALLs lacking PKCθ show improved LIC activity whereas enforced PKCθ expression in both mouse and human primary T-ALLs compromised LIC activity. We also demonstrate that PKCθ is positively regulated by RUNX1, and that NOTCH1, which is frequently activated by mutation in T-ALL 4 – 6 and required for LIC activity in both mouse and human models 7 , 8 , downregulates PKCθ and ROS via a novel pathway involving induction of RUNX3 and subsequent repression of RUNX1. These results reveal key functional roles for PKCθ and ROS in T-ALL and suggest that aggressive biological behavior in vivo could be limited by therapeutic strategies that promote PKCθ expression/activity or ROS accumulation.