Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions. Display omitted •Results of screens to identify drivers of hematopoietic differentiation•Perturbing nucleotide metabolism and DNA replication promotes differentiation•Replication stress activates primed regulatory loci to induce lineage programs•Canonical signaling and DNA damage responses to replication stress are not required Do, Hsu et al. find that in non-transformed and transformed hematopoietic systems, perturbing nucleotide metabolism and DNA replication drives cells toward a more differentiated state and that replication stress activates primed chromatin to induce lineage-specifying programs, even when oncogenic differentiation blockades remain intact.