T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease. The PHF6 gene is frequently targeted by loss-of-function mutations or deletions, with the highest prevalence in TLX1 or TLX3 rearranged T-ALLs. We aim to gain insights into the role of PHF6 during normal thymocyte development and malignant transformation. To this end, transcriptome wide perturbation effects were measured following PHF6 knock down in the PHF6 wild-type T-ALL cell lines Jurkat and ALL-SIL. In parallel, gene expression profiles were established from cord blood CD34+ progenitor T-cells, cultured for short-term on an OP9-DL1 feeder layer, with stable knockdown of PHF6. Modulation of PHF6 expression induced robust and broad transcriptional regulatory effects of PHF6 including on IL7R expression. IL7R encodes a cytokine receptor critically involved in normal T-cell development and acts as a bona fide oncogene in subsets of T-ALLs. Using the in vitro differentiation assay for all lineages starting from CD34+ cells we also identified a broader role for PHF6 beyond thymocyte differentiation acting as a master regulator of early hematopoietic differentiation along the T-cell, B-cell, myeloid and NK-cell lineages (Durinck et al., in preparation). To further test this in vivo, we performed phf6 gene inactivation by injection of gRNAs and Cas9 protein into the one-cell stage zebrafish embryos yielding varying out-of-frame in/dels. In addition, we used a TALEN based PHF6 knock out zebrafish line (Moore et al., Plos One, 2012). Upon phf6 knockdown accelerated T-cell maturation was seen in support of our in vitro data in human cells. Interestingly, transcriptional changes in the phf6 knock out lines were noted for lmo2 and notch1, both key players in zebrafish hematopoiesis. Using optimized of RT-qPCR and RNA sequencing on sorted lymphocytes we are currently evaluating the transcriptional effects of phf6 down regulation during lymphocyte development. We will present findings on whole kidney marrow tissue for changes in hematopoietic population distribution in wild type versus phf6 knock out zebrafish. In conclusion, we were able to show for the first time a conclusive role for PHF6 in hematopoiesis using in vitro and in vivo modeling in human and zebrafish respectively.