The Notch signaling pathway plays important roles in cell-fate determination during embryonic development and adult life. In this study, we focus on the role of Notch signaling in governing cell-fate choices in human embryonic stem cells (hESCs). Using genetic and pharmacological approaches, we achieved both blockade and conditional activation of Notch signaling in several hESC lines. We report here that activation of Notch signaling is required for undifferentiated hESCs to form the progeny of all three embryonic germ layers, but not trophoblast cells. In addition, transient Notch signaling pathway activation enhanced generation of hematopoietic cells from committed hESCs. These new insights into the roles of Notch in hESC-fate determination may help to efficiently direct hESC differentiation into therapeutically relevant cell types.