The profound intestinal epithelial defects in the newborn epidermal growth factor receptor (EGFR) knockout mouse suggests that EGFR signaling plays important roles in embryonic gut development. Herein, we further elucidated the function of EGFR signaling on early embryonic gut development by comparing the effects of 1–10 ng/mL of exogenous epidermal growth factor (EGF) or 10–25 μM of the tyrphostin 3, 4, 5 trihydroxybenzene malononitrile, a specific inhibitor of EGFR tyrosine kinase, on intact E12 Swiss-Webster mouse midgut grown in chemically defined organ culture using Fitton-Jackson BGJb medium for 4 or 6 d. Intestinal development during culture was assayed by morphometry, histology, reverse transcription/competitive PCR for villin and intestinal fatty acid binding protein mRNA, and immunohistochemistry for epithelial proliferative markers. During organ culture, control specimens grew in length, developed smooth muscle, simple columnar epithelial and goblet cell phenotypes, showed early villus formation in the proximal intestine, and increased expression of villin and intestinal fatty acid binding protein mRNA. EGF failed to significantly alter small intestinal lengthening, whereas EGF 10 ng/mL inhibited colonic length growth. Tyrphostin 25 μM resulted in regional losses of stromal and smooth muscle cells in the small intestine and absent colonic goblet cells. In controls, cellular proliferation initially occurred throughout the small intestinal epithelium but became increasingly localized to the intervillus crypt regions. This sequestration of epithelial proliferation into crypts was much more apparent in EGF-treated versus tyrphostin-treated specimens. EGFR activation, therefore, appears to accelerate the maturation rate of goblet cells and the differential crypt/villus proliferation pattern in early embryonic mouse gut.