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Somatic mutations in genes encoding factors involved in pre-mRNA splicing (i.e., spliceosome genes) have been identified in more than half of myelodysplastic syndrome (MDS) patients. We are interested in the role of U2AF1, a spliceosome gene mutated in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1 have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type (WT) U2AF1 in normal hematopoiesis has not been studied. A complete understanding of the role of WT U2AF1 on hematopoiesis and RNA splicing is critical to our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of WT U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse. Homozygous embryonic deletion of U2af1 using Vav1-Cre was lethal, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wt or Mx1-Cre/U2af1wt/wt into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1 deletion, homozygous U2af1 KO mice, but not other genotypes, became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KO mice compared to all other genotypes (p ≤ 0.001, n=5). Deletion of U2af1 also led to rapid bone marrow failure and a reduction in bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (p ≤ 0.001, n=5). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and WT congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cells (HSCs). As early as 10 days, and up to 4 months following Mx1-Cre-induction, we observed a significant decrease in white blood cell count chimerism and complete loss of homozygous U2af1 KO HSCs, neutrophils and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). Collectively, the data indicate that normal hematopoiesis is dependent on U2af1 expression. In ongoing studies, we crossed the U2af1 KO mouse to existing transgenic mutant U2AF1-expressing mice to determine if mutant U2AF1 expression is sufficient to rescue cell survival and normal hematopoiesis when endogenous WT U2af1 is deleted. If U2AF1 mutant cells are vulnerable to loss of the WT U2AF1 allele, then selectively targeting the WT U2AF1 allele in heterozygous mutant cells could be a novel therapeutic strategy. Citation Format: Brian A. Wadugu, Amanda Heard, Joseph Bradley, Matthew Ndonwi, Matthew J. Walter. U2af1, a spliceosome gene commonly mutated in MDS, is required for hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5112.