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Notch signaling has been shown over the past few decades to play fundamental roles in a plethora of developmental processes in an evolutionarily conserved fashion. Notch-mediated cell-to-cell signaling is involved in many aspects of embryonic development and control of tissue homeostasis in a variety of adult tissues, and regulates stem cell maintenance, cell differentiation and cellular homeostasis. The focus of this Review is the role of Notch signaling in stem cells, comparing insights from flies, fish and mice to highlight similarities, as well as differences, between species, tissues and stem cell compartments.