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E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their in vitro differentiation was incomplete. In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment., Author Summary Disease may be due to either excess of undesirable cells, like in cancer or autoimmune disease, or by progressive loss of vital cells. The latter, for instance, causes neurodegenerative diseases such as Alzheimer’s disease. Stem-cell based therapy holds great potential to cure devastating diseases with cell loss or dysfunctionality, because stem cells have the capacity to form any given cell type of the body. Recent advances in the field allow to obtain stem cells from virtually every patient. These stem cells could then be instructed to form the desired cells that can be reintroduced to cure the patient. Before such therapies are suited for the clinic, we first need comprehensive knowledge of the molecular mechanisms that underlie cell fate decisions. Here, we scrutinize the role of a junctional protein, called p120ctn, in both stem cells and lineage-committed cells. Importantly, this key protein has a modular structure, and each of its segments has different interaction partners and biological functions. We deleted p120ctn specifically in stem cells, and reintroduced several p120ctn mutants that lack specific protein segments. As such, we could unravel the exact molecular interaction that is required for p120ctn to drive the differentiation of stem cells towards primitive endoderm.