Here, using metabolic assays, immunoblotting, flow cytometry analyses, and siRNA-mediated gene silencing in leukemia cell lines, we show that AICAr-mediated differentiation was independent of the known metabolic effects of AMPK, including glucose consumption, but instead depends on the activation of the DNA damage–associated enzyme checkpoint kinase 1 (Chk1) induced by pyrimidine depletion. Here, CHEK1 is linked to leukemia.