Human embryonic stem cells (hESCs) provide an essential tool to investigate early human development, study disease pathogenesis, and examine therapeutic interventions. Adenomatous polyposis coli (APC) is a negative regulator of Wnt/β-catenin signaling, implicated in the majority of sporadic colorectal cancers (CRC) and in the autosomal dominant inherited syndrome familial adenomatous polyposis (FAP). Studies into the role of Wnt/β-catenin signaling in hESCs arrived at conflicting results, due at least in part to variations in culture conditions and the use of external inhibitors and agonists. Here, we directly targeted APC in hESCs carrying a germline APC mutation, derived from affected blastocysts following preimplantation genetic diagnosis (PGD) for FAP, in order to answer open questions regarding the role of APC in regulating pluripotency and differentiation potential of hESCs. Using CRISPR/Cas9 we generated second hit APC mutations in FAP-hESCs. Despite high CRISPR/Cas9 targeting efficiency and the successful isolation of many clones, none of the isolated clones carried a loss of function mutation in the WT-APC allele. Using a fluorescent β-catenin reporter and analysis of mutated-allele frequencies in the APC locus, we show that APC double mutant hESCs robustly activate Wnt/β-catenin signaling that results in rapid differentiation to endodermal and mesodermal lineages. Here we provide direct evidence for a strict requirement for constant β-catenin degradation through the APC destruction complex in order to maintain pluripotency, highlighting a fundamental role for APC in self-renewal of hESCs. SIGNIFICANCE STATEMENT: Studying Human embryonic stem cell (hESC) self-renewal is essential to instruct efforts into their maintenance, and differentiation. Although Wnt/ß-catenin signaling has a pivotal roles in regulating cell fate in many tissues, its role in controlling hESC differentiation and in regulating pluripotency remains ambiguous. Here, we show that Wnt/ß-catenin signaling activity is low in undifferentiated hESCs, and that this low activity is at least partly due to active repression by the tumor suppressor APC. We further demonstrate that APC has a pivotal role in hESC self-renewal by inhibiting Wnt/ß-catenin induced differentiation, and that upon its loss cells rapidly differentiate into mesendoderm. © AlphaMed Press 2019.