Abstract Body: Pulmonary arterial hypertension (PAH) is a progressive disorder marked by vasculopathy in the distal pulmonary vessels, resulting in increased vascular resistance, subsequent right ventricular hypertrophy, and eventual failure. Current treatments provide symptom relief, but the lack of a definitive cure emphasizes the need for innovative therapies targeting the underlying cellular and molecular mechanisms. Sox17 is one of these targets that stands out as crucial for the proper development of pulmonary vasculature and endothelial regeneration after vascular injury, given that its loss-of-function mutations are linked to more severe and early-onset PAH. During fetal development, SOX17 directly binds to Runx1 (a downstream target), suppressing the formation of hematopoietic cells during the endothelial-to-hematopoietic transition—a pivotal process for maintaining arterial differentiation and identity. Our previous studies revealed that Sugen/Hypoxia-induced PAH could be prevented by tissue-specific deletion of Runx1 in the endothelium, underscoring its critical role in PAH development. Therefore, we propose that the lack of repression of Runx1 by endothelial SOX17 contributes to the development of PAH through impaired arterial endothelial differentiation and we aim to investigate how a compromised SOX17/Runx1 axis influences the development of PAH using human pulmonary arterial cells (HPAEC). This study reveals that the overexpression of Runx1 in HPAEC induces significant alterations in cellular properties, including loss of arterial endothelial features and a significant alteration in gene expression, shifting the cells from an endothelial to a hematopoietic phenotype, characterized by a decrease in the expression of vWF and ICAM-1 and an increase in CBFB, CD14, and CD27. Furthermore, HPAEC cells overexpressing Runx1 exhibited reduced endothelial sprouting and abnormal angiogenesis in an in vitro tube formation assay, noticeable morphological changes, and a decrease in the uptake of low-density lipoprotein (LDL). Our findings emphasize the importance of maintaining low Runx1 levels in HPAECs to preserve endothelial cell identity, which may be disrupted by decreased SOX17 expression in PAH patients with SOX17 mutations. It also sheds light on Runx1's roles in endothelial cell biology and angiogenic processes that contribute to PAH pathogenesis, as well as the therapeutic potential of targeting the SOX17/Runx1 axis as a promising approach for PAH treatment.