Arsenic is a ubiquitous toxic metalloid, with over 150 million people exposed to arsenic concentrations above the current 10ppb drinking water standard through contaminated food and water. Arsenic is a known developmental toxicant as neuronal and muscle development are disrupted following arsenic exposure during embryogenesis. In this study, murine embryonic stem cells were chronically exposed to 0.1μM (7.5ppb) arsenic for 32weeks. RNA sequencing showed that the Hippo signaling pathway, which is involved in embryonic development and pluripotency maintenance, is impaired following arsenic exposure. Thus, temporal changes in the Hippo pathway's core components and its downstream target genes Ctgf and c-Myc were investigated. Protein expression of the pathway's main effector YAP in its active form was significantly upregulated by 3.7-fold in arsenic-exposed cells at week 8, while protein expression of inactive phosphorylated YAP was significantly downregulated by 2.5- and 2-fold at weeks 8 and 16. Exposure to arsenic significantly increased the ratio between nuclear and cytoplasmic YAP by 1.9-fold at weeks 16 and 28. The ratio between nuclear and cytoplasmic transcriptional enhancer factor domain was similarly increased in arsenic-treated samples by 3.4- and 1.6-fold at weeks 16 and 28, respectively. Levels of Ctgf and c-Myc were also upregulated following arsenic exposure. These results suggest that chronic exposure to an environmentally relevant arsenic concentration might hinder cellular differentiation and maintain pluripotency through the impairment of the Hippo signaling pathway resulting in increased YAP activation.