Self-renewing hematopoietic stem cells (HSCs) generate all blood cells and give rise to long-term reconstitution of the hematopoietic system after transplantation, but the molecular mechanisms that specifically regulate HSCs remain poorly defined. Here, we found that HSCs displayed a distinct messenger RNA alternative splicing pattern and preferentially expressed Rbfox2, an alternative splicing regulator, compared with multipotent progenitors (MPPs). Deletion of Rbfox2 from the hematopoietic compartment specifically depleted HSCs, but not progenitors in the adult bone marrow. Rbfox1 did not function redundantly with Rbfox2 in HSCs. Mechanistically, Rbfox2 loss led to proteostasis stress, including increased protein synthesis rate and accumulated misfolded/unfolded protein contents, in HSCs, but not in progenitors. Small molecules that restore proteostasis rescued HSC defects in Rbfox2-deficient mice. Our work thus reveals that HSCs, but not progenitors, selectively rely on Rbfox2 for their self-renewal and maintenance.