The molecular mechanism underlying the development of Barrett's esophagus (BE), the precursor to esophageal adenocarcinoma, remains unknown. Our previous work implicated sonic hedgehog (Shh) signaling as a possible driver of BE and suggested that bone morphogenetic protein 4 (Bmp4) and Sox9 were downstream mediators. We have utilized a novel in vivo tissue reconstitution model to investigate the relative roles of Bmp4 and Sox9 in driving metaplasia. Epithelia reconstituted from squamous epithelial cells or empty vector-transduced cells had a stratified squamous phenotype, reminiscent of normal esophagus. Expression of Bmp4 in the stromal compartment activated signaling in the epithelium but did not alter the squamous phenotype. In contrast, expression of Sox9 in squamous epithelial cells induced formation of columnar-like epithelium with expression of the columnar differentiation marker cytokeratin 8 and the intestinal-specific glycoprotein A33. In patient tissue, A33 protein was expressed specifically in BE, but not in normal esophagus. Expression of Cdx2, another putative driver of BE, alone had no effect on reconstitution of a squamous epithelium. Furthermore, epithelium coexpressing Cdx2 and Sox9 had a phenotype similar to epithelium expressing Sox9 alone. Our results demonstrate that Sox9 is sufficient to drive columnar differentiation of squamous epithelium and expression of an intestinal differentiation marker, reminiscent of BE. These data suggest that Shh-mediated expression of Sox9 may be an important early event in the development of BE and that the potential for inhibitors of the hedgehog pathway to be used in the treatment of BE and/or esophageal adenocarcinoma could be tested in the near future.