Abstract Plant Home domain Finger 6 (PHF6) is a tumor suppressor of unknown function for blood malignancies such as T-cell Acute Lymphoblastic Leukemia (T-ALL), Acute Myeloid leukemia (AML), and Chronic Myeloid Leukemia (CML). PHF6 contains two zinc finger-like PH domains and interacts with the Nucleosome Remodeling and Deacetylation (NuRD) complex, suggesting a role in chromatin remodeling. Although PHF6 loss-of-function mutations are found in nearly 40% of T-ALL patients, little is known about how PHF6 mutations contribute to blood development and leukemogenesis. To understand the function of PHF6, beginning with its role in hematopoiesis, we have undertaken developmental studies in zebrafish to discover how loss of phf6 affects blood development and to determine which pathways are regulated by phf6. Zebrafish will be used to study hematopoiesis due to the remarkable conservation of molecular pathways that regulate blood development, genetic tractability, and ability to observe embryonic development over a short window of time. RNA in situ hybridization studies of zebrafish embryos showed that phf6 is expressed broadly during zebrafish development, and especially in the dorsal aorta, a site analogous to the aorta-gonad-mesonephros (AGM) in mammals, from which hematopoietic stem cells (HSCs) arise. Further, phf6 is highly expressed in lymphocytes of adult zebrafish, reminiscent of the expression patterns found in human and mouse. To determine the effect of phf6 loss on hematopoiesis, phf6 expression was knocked down by morpholino injection. We find that phf6 morphants have increased numbers of HSCs by RNA in situ hybridization of runx1/cmyb in the AGM and caudal hematopoietic tissue ((CHT) analogous to mammalian fetal liver), sites of HSC emergence and migration. Later in development, phf6 morphants demonstrate increased lymphocytes by RNA in situ hybridization of rag1 at the thymus. Similar phenotypes were observed in homozygous phf6-null mutants generated by TALEN-mediated knockout. Phf6-null mutant zebrafish survive to Mendelian ratios and are fertile as adults. In total, we found a new role for phf6 in regulation of HSC formation. Citation Format: Finola E. Moore, Virginie Esain, Riadh Lobbardi, Jessica S. Blackburn, Trista E. North, David M. Langenau. Role for the tumor suppressor phf6 in hematopoiesis. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A33.