Recurrent, heterozygous, somatic mutations in components of the mRNA spliceosome complex were recently identified in over 60% of myelodysplastic syndrome (MDS) patients. Splicing factor mutations are thought to be founding mutations in MDS based on their allele fraction at diagnosis. Splicing factor 3b Subunit 1 (SF3B1) is the most frequently mutated splicing factor in MDS. SF3B1 mutations are highly associated (70 – 85% of cases) with refractory anemia with ring sideroblasts (RARS), a morphologic subtype of MDS characterized by the presence of erythroid precursors with perinuclear iron-laden mitochondria in the bone marrow. The pathophysiological role of SF3B1 mutations in MDS has yet to be elucidated. To explore the biology of SF3B1 mutations, we generated a heterozygous conditional knock-in mouse model of the most common SF3B1 mutation, K700E. Heterozygous conditional knock-in of Sf3b1K700E leads to a progressive macrocytic anemia, with normal absolute neutrophil and platelet counts. Over the course of 15 months, mutant mice developed a statistically significant macrocytic anemia (hemoglobin of 11.4 g/dL vs. 14 g/dL, p = 0.004; MCV of 63.1 fL vs. 58.4 fL, p = 0.008) associated with elevated plasma erythropoietin levels (257.5 pg/mL vs. 101 pg/mL, p = 0.0016). Analysis of hematopoietic stem and progenitor cells at 12 and 65 weeks after induction showed a similar percentage of stem (LT-HSC, ST-HSC, MPP, LSK) and progenitor (LK, CMP, GMP, MEP, pre CFU-E) cells in Sf3b1K700E and wild-type animals. Histopathologic analysis revealed no significant difference in spleen weights, but increased erythroid islands in the red pulp of mutant animal spleens; suggestive of ineffective erythroid maturation. Sf3b1K700E animals have a normocellular bone marrow with rare ring sideroblasts. No ring sideroblasts were identified in wild-type controls. No overt hematological malignancies were identified during the observation period, however two mutant animals succumbed to significant anemia (2 of 11, 18%) compared to zero deaths in the wild-type controls. To further characterize the erythroid-specific phenotype observed in Sf3b1K700E mice, mutant and wild-type animals were treated with phenylhydrazine, a drug that induces intravascular hemolysis. Sf3b1K700E mice had a more rapid onset of anemia and a higher reticulocytosis during count recovery compared to wild-type controls. Analysis of the bone marrow and spleens was notable for a higher percentage of immature erythroid precursors (R2/basophilic erythroblasts) and a lower percentage of more mature erythroid precursors (R4/orthochromatophilic erythroblasts) in mutant animals, consistent with impaired erythroid maturation. An in vitro erythroid differentiation assay using purified ckit + progenitor cells from Sf3b1K700E mice yielded significantly fewer erythroblasts (p = 0.0226) when compared to cells from wild type mice due to a statistically significant increase in the percentage of mutant cells in G0 (p=0.018). Similarly, noncompetitive transplantation assays highlighted the cell intrinsic nature of these erythroid-specific findings, as mutant cells did not show a defect in repopulating recipients, however Sf3b1K700E recipients developed a progressive macrocytic anemia. Competitive transplantation assays demonstrated a competitive disadvantage in Sf3b1K700E hematopoietic stem cells. Engraftment was lower in Sf3b1K700E compared to wild-type recipients 4 weeks (33.9% vs. 54.4%, p = 0.002) and 16 weeks (29% vs. 62.4%, p = 0.0013) after transplantation. These findings are consistent with the fact that RARS patients have a lower risk of progression to acute myeloid leukemia compared with other MDS subtypes. Taken together, our results demonstrate that heterozygous mutations in Sf3b1 lead to aberrant erythroid maturation and ineffective hematopoiesis in mice. These findings are consistent with the clinical picture seen in RARS patients. The results from the competitive transplantation studies may be consistent with the more favorable prognosis seen in patients with RARS, as our data suggest that additional genetic or epigenetic alterations must be acquired in SF3B1K700E cells to facilitate the development of clonal dominance. Disclosures No relevant conflicts of interest to declare.