During development, major metabolic changes occur as cells become more specialized within a lineage. In the case of skeletal muscle, differentiation is accompanied by a switch from a glycolytic proliferative progenitor state to an oxidative postmitotic differentiated state. Such changes require extensive mitochondrial biogenesis leading to increased reactive oxygen species (ROS) production that needs to be balanced by an antioxidant system. Our analysis of double conditional Pitx2/3 mouse mutants, both in vivo during fetal myogenesis and ex vivo in primary muscle cell cultures, reveals excessive upregulation of ROS levels leading to DNA damage and apoptosis of differentiating cells. This is a consequence of downregulation of Nrf1 and genes for antioxidant enzymes, direct targets of Pitx2/3, leading to decreased expression of antioxidant enzymes, as well as impairment of mitochondrial function. Our analysis identifies Pitx2 and Pitx3 as key regulators of the intracellular redox state preventing DNA damage as cells undergo differentiation.