Prion protein (PrPC), is a glycoprotein that is expressed on the cell surface. The current study examines the role of PrPC in early human embryogenesis using human embryonic stem cells (hESCs) and tetracycline-regulated lentiviral vectors that up-regulate or suppresses PrPC expression. Here, we show that expression of PrPC in pluripotent hESCs cultured under self-renewal conditions induced cell differentiation toward lineages of three germ layers. Silencing of PrPC in hESCs undergoing spontaneous differentiation altered the dynamics of the cell cycle and changed the balance between the lineages of the three germ layers, where differentiation toward ectodermal lineages was suppressed. Moreover, over-expression of PrPC in hESCs undergoing spontaneous differentiation inhibited differentiation toward lineages of all three germ layers and helped to preserve high proliferation activity. These results illustrate that PrPC is involved in key activities that dictate the status of hESCs including regulation of cell cycle dynamics, controlling the switch between self-renewal and differentiation, and determining the fate of hESCs differentiation. This study suggests that PrPC is at the crossroads of several signaling pathways that regulate the switch between preservation of or departure from the self-renewal state, control cell proliferation activity, and define stem cell fate.