The stem/progenitor cells of the developing intestine are biologically distinct from their adult counterparts. Here, we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor delta-like protein-1 (DLK1). mRNA-seq analyses of intestinal mesenchymal cells (IMCs) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel coculture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR, and immunocytochemistry. The intestinal phenotypes of littermate Dlk1þ / þ and Dlk1-/- mice were compared using immunohistochemistry. Using transcriptomic analyses, we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth, and shifted to the intestinal epithelium in adulthood. In coculture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knockdown in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1-/mice had restricted Ki67þ cells in the villi base and increased secretory lineage cells compared with Dlk1þ / þ embryos. Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine. NEW & NOTEWORTHY Using a novel coculture system, transcriptomics, and transgenic mice, we investigated differential molecular signaling between the intestinal epithelium and mesenchyme during development and in the adult. We show that the Notch pathway factor delta-like protein-1 (DLK1) is stromally produced during development and uncover a new role for DLK1 in the regulation of intestinal epithelial stem/precursor expansion and differentiation to secretory lineages.