Hematopoietic stem and progenitor cells (HSPCs) sustain lifelong hematopoiesis. Mutations of pre-mRNA splicing machinery, especially splicing factor 3b, subunit 1 (SF3B1), are early lesions found in malignancies arising from HSPC dysfunction. However, why splicing factor deficits contribute to HSPC defects remains incompletely understood. Using zebrafish, we show that HSPC formation in sf3b1 homozygous mutants is dependent on STAT3 activation. Clinically, mutations in SF3B1 are heterozygous; thus, we explored if targeting STAT3 could be a vulnerability in these cells. We show that SF3B1 heterozygosity confers heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Cells carrying mutations in other splicing factors or treated with splicing modulators are also more sensitive to STAT3 inhibition. Mechanistically, we illustrate that STAT3 inhibition exacerbates aberrant splicing in SF3B1 mutant cells. Our findings reveal a conserved vulnerability of splicing factor mutant HSPCs that could allow for their selective targeting in hematologic malignancies. •Sf3b1 loss-of-function HSPCs are highly dependent on STAT3 signaling•SF3B1 heterozygosity confers heightened sensitivity to STAT3 inhibition•STAT3 inhibition aggravates splicing defects in SF3B1 mutant cells Potts et al. show that STAT3 inhibition is a conserved vulnerability for splicing factor heterozygous mutant hematopoietic cells due to its impact on splicing. These findings suggest that STAT3 inhibitors could be used alone or in combination with splicing modulators to selectively ablate splicing-defective cells in blood cancers.