Significance Somatic mutations in some splicing factor genes are frequently found in myelodysplastic syndromes (MDS) and MDS-related acute myeloid leukemia (AML), blood cancers with few effective treatment options. However, the pathophysiological effects of these mutations remain poorly characterized. Here, we report the establishment of mouse models to study a common splicing factor mutation, U2AF1 (S34F). Production of the mutant protein in the murine hematopoietic compartment disrupts hematopoiesis in ways resembling human MDS. We further identified deletion of the Runx1 gene and other known oncogenic mutations as changes that might collaborate with U2af1 (S34F) to give rise to frank AML in mice. However, the U2af1 (S34F) mutation was absent in two of the three AML cases, raising the possibility that this mutant protein plays a dispensable role in tumor maintenance.