The placenta is the connecting organ between an embryo and its mother and it fulfils a multitude of tasks that later in life are accomplished by a number of individual organs. Despite its pivotal function during pregnancy and its postulated involvement in the pathogenesis of major gestational disorders such as pre-eclampsia, miscarriage or intrauterine growth restriction it is a rather neglected organ. Placental functions involve nutrient and oxygen supply of the embryo as well as waste disposal. To realise that, formation of an accurate foetal-maternal interface and proper trophoblast invasion into uterine tissue with subsequent vascular remodelling is essential. Nevertheless, molecular signalling pathways controlling this tightly regulated process are still poorly elucidated. In this study I provide evidence that the canonical Notch signalling pathway, acting through its central transcription factor RBPJkappa, plays a major role in controlling proliferation and differentiation of first trimester primary trophoblasts and villous explant cultures. RBPJkappa and its co-activators MAML1-3 were detected in human villous cytotrophoblasts (CTBs) and extravillous trophoblasts (EVTs) of tissue sections using immunofluorescence. Basic Notch activity was measured in isolated CTBs after transfection with an RBPJκ-dependent luciferase reporter. However, RBPJkappa expression, transcript levels of its target gene HES1 and reporter activity declined during in vitro formation of EVTs. SiRNA-mediated gene knock-down of RBPJkappa extinguished Notch reporter expression and enhanced proliferation of both, isolated CTBs and cell column trophoblasts in villous explant cultures, promoting an increased outgrowth of the latter. Accordingly, markers of the differentiated EVT, i.e. ADAM12, ITGA1 and T-cell factor 4 were upregulated upon gene silencing. Notably, motility and apoptosis were not affected. These data suggest a Notch-dependent control of cell column trophoblast progenitors that could represents a mechanism to prevent exaggerated placental growth by balancing rates of differentiation and proliferation.