Abstract Loss-of-function mutations of EZH2, the enzymatic component of PRC2, have been associated with poor outcome and chemotherapy resistance in T-cell acute lymphoblastic leukemia (T-ALL). Using isogenic T-ALL cells, with and without CRISPR/Cas9–induced EZH2-inactivating mutations, we performed a cell-based synthetic lethal drug screen. EZH2-deficient cells exhibited increased sensitivity to structurally diverse inhibitors of CHK1, an interaction that could be validated genetically. Furthermore, small-molecule inhibition of CHK1 had efficacy in delaying tumor progression in isogenic EZH2-deficient, but not EZH2 wild-type, T-ALL cells in vivo, as well as in a primary cell model of PRC2-mutant ALL. Mechanistically, EZH2 deficiency resulted in a gene-expression signature of immature T-ALL cells, marked transcriptional upregulation of MYCN, increased replication stress, and enhanced dependency on CHK1 for cell survival. Finally, we demonstrate this phenotype is mediated through derepression of a distal PRC2-regulated MYCN enhancer. In conclusion, we highlight a novel and clinically exploitable pathway in high-risk EZH2-mutated T-ALL. Significance: Loss-of-function mutations of PRC2 genes are associated with chemotherapy resistance in T-ALL, yet no specific therapy for this aggressive subtype is currently clinically available. Our work demonstrates that loss of EZH2 activity leads to MYCN-driven replication stress, resulting in increased sensitivity to CHK1 inhibition, a finding with immediate clinical relevance. This article is highlighted in the In This Issue feature, p. 890