SummaryThe circadian clock is a molecular pacemaker that produces 24-hr physiological cycles known as circadian rhythms. How the clock regulates stem cells is an emerging area of research with many outstanding questions. We tested clock function in vivo at the single cell resolution in the Drosophila intestine, a tissue that is exquisitely sensitive to environmental cues and has circadian rhythms in regeneration. Our results indicate that circadian clocks function in intestinal stem cells and enterocytes but are downregulated during enteroendocrine cell differentiation. Drosophila intestinal cells are principally synchronized by the photoperiod, but intestinal stem cell clocks are highly responsive to signaling pathways that comprise their niche, and we find that the Wnt and Hippo signaling pathways positively regulate stem cell circadian clock function. These data reveal that intestinal stem cell circadian rhythms are regulated by cellular signaling and provide insight as to how clocks may be altered during physiological changes such as regeneration and aging.