The ETO2-GLIS2 fusion is associated with young and aggressive pediatric acute megakaryoblastic leukemia (AMKL) and is thought to arise in utero. To investigate ETO2-GLIS2 consequences in human hematopoietic cells and overcome the difficulty to access appropriate fetal stages, we engineered induced pluripotent stem cells (iPSC) to obtain hematopoietic-specific ETO2-GLIS2 expression and analyzed in vitro hematopoietic differentiation of IPSC toward the megakaryocytic lineage, which presents common features with human hematopoiesis during early development. As compared to controls, ETO2-GLIS2 expression induced an increased proportion of CD41+42+ megakaryocytes at several differentiation timepoints and generated a CD41low42low population that is absent in controls. In addition, ETO2-GLIS2 enhanced proliferation and self-renewal capacities in methylcellulose assays. To understand the molecular consequences of ETO2-GLIS2 expression in human progenitors, we next performed RNA sequencing on flow purified CD41+42+ megakaryocytes and the abnormal CD41low42low population. Compared to wild-type CD41+42+ profiles, ETO2-GLIS2-expressing CD41+42+ profiles were enriched for several ETO2-GLIS2 AMKL patients blasts signatures and an ETO2-GLIS2-dependent enhancer-associated genes signature. Importantly, these signatures were even more enriched in the ETO2-GLIS2-expressing CD41low42low suggesting that this abnormal population more closely mimic the leukemic blasts found in patients. Of note, expression of ERG and GATA1, two master hematopoietic transcription factors, were not deregulated to the same extent as in patients’ blasts. Together, this human ETO2-GLIS2 iPSC model recapitulates differentiation alterations, increased selfrenewal and transcriptional signatures observed in human AMKL and should therefore represent an interesting platform to perform future molecular and preclinical investigations. De novo pediatric acute megakaryoblastic leukemia (AMKL) is characterized by fusion oncogenes. 1, 2 Murine transgenic models have been reported for some fusions, including OTT-MAL and MN1-FLI1. 3, 4 The CBFA2T3-GLIS2 (ETO2-GLIS2) fusion, present in 20% to 30% of de novo pediatric AMKL, is associated