Immortal spheroids were generated from fetal mouse intestine using the culture system initially developed to culture organoids from adult intestinal epithelium. Spheroid proportion progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Like organoids, spheroids show Wnt-dependent indefinite self-renewing properties but display a poorly differentiated phenotype reminiscent of incompletely caudalized progenitors. The spheroid transcriptome is strikingly different from that of adult intestinal stem cells, with minimal overlap of Wnt target gene expression. The receptor LGR4, but not LGR5, is essential for their growth. Trop2/Tacstd2 and Cnx43/Gja1, two markers highly enriched in spheroids, are expressed throughout the embryonic-day-14 intestinal epithelium. Comparison of in utero and neonatal lineage tracing using Cnx43-CreER and Lgr5-CreERT2 mice identified spheroid-generating cells as developmental progenitors involved in generation of the prenatal intestinal epithelium. Ex vivo, spheroid cells have the potential to differentiate into organoids, qualifying as a fetal type of intestinal stem cell. Display omitted •Fetal and adult intestinal epithelia are generated from two kinds of progenitor cells•Trop2 and Cnx43 are markers of fetal intestinal progenitors•Ex vivo, fetal intestinal progenitors generate immortal undifferentiated spheroids•Spheroids have the ability to convert to differentiated Lgr5-positive organoids Vassart, Garcia, and colleagues now use lineage-tracing experiments to demonstrate that definitive mouse intestinal epithelium is generated in two waves depending on different sets of progenitors. The authors show that immortal, undifferentiated spheroids can be obtained from culturing fetal intestinal epithelium under conditions yielding differentiated organoids from adult intestine. Spheroid cells display a transcriptome strikingly different from that of adult intestinal stem cells. The cells correspond to progenitors “frozen” in a state preceding differentiation into fetal intestinal epithelium and generation of adult stem cells.