NOTCH signaling is a key regulator involved in maintaining intestinal stem cell (ISC) homeostasis and for balancing differentiation. Using single-cell transcriptomics, we observed that OLFM4, a NOTCH target gene present in ISCs, is first expressed at 13 weeks postconception in the developing human intestine and increases over time. This led us to hypothesize that the requirement for NOTCH signaling is acquired across human development. To test this, we established a series of epithelium-only organoids (enteroids) from different developmental stages and used g-secretase inhibitors (dibenzazepine [DBZ] or DAPT) to functionally block NOTCH signaling. Usingquantitative enteroid-formingassays,weobservedadecreaseinenteroidformingefficiencyinresponsetog-secretaseinhibitionas development progress. When DBZwasaddedtoculturesand maintained duringroutine passaging, enteroids isolated from tissue before 20 weeks had higher recovery rates following single-cell serial passaging. Finally, bulk RNA sequencing (RNA-seq) analysis 1 day and 3daysafter DBZtreatmentshowedmajordifferencesin the transcriptional changes between developingor adult enteroids. Collectively, these data suggest that ISC dependence on NOTCH signaling increases as the human intestine matures.