Epigenetic regulation exerts a crucial role in maintaining the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs), thereby sustaining normal hematopoiesis. Although emerging evidence has demonstrated the contribution of RNA N4-acetylcytidine (ac4C) modification and its catalytic enzyme Nat10 to numerous biological processes, particularly tumorigenesis, the function of Nat10 in normal hematopoiesis remain unexplored. In this study, we characterize the function of Nat10 in hematopoietic stem and progenitor cells (HSPCs) using low input proteomics and reveal a key role of Nat10 in the maintenance of HSCs.