BACKGROUND AND AIMS: Metastatic cancers arise from a decades-long succession of increasingly virulent precursor lesions, each of which represents prospective targets for therapeutic intervention. This evolutionary process has been particularly vivid in esophageal adenocarcinoma (EAC), as this cancer and associated precursor lesions, including Barrett's esophagus (BE), low-grade dysplasia (LGD), and high-grade dysplasia (HGD), co-exist in an accessible, two-dimensional pattern in esophageal mucosa. Given the durability of these precursor lesions, it is likely that they, like EAC, rely on stem cells for their regenerative growth. To assess the role of stem cells in the evolution of EAC, we apply technology that selectively clones stem cells from the gastrointestinal tract to patient-matched endoscopic biopsies from each of the precursor lesions implicated in EAC. METHODS: Histologically validated, endoscopic biopsy series including EAC, HGD, LGD, BE, and normal esophageal mucosa were obtained from patients presenting with EAC. Rare (1:1,000) cells from each of these lesions proved clonogenic and were assessed by in vitro differentiation, tumorigenicity in mice, and by molecular genetics. RESULTS: Each of the lesions in the evolution of EAC possess a discrete set of clonogenic cells marked by immaturity, enormous proliferative potential, and lesion-specific differentiation fate. DNA sequencing of these clones reveal intralesional heterogeneity and clonal resolution of the mutation progression within a given patient from BE, LGD, HGD, and EAC. High-throughput chemical screens against BE stem cells reveal drug combinations that are similarly effective against stem cells of LDG, HGD, and EAC. CONCLUSIONS: All lesions in the evolution of EAC possess discrete populations of stem cells that are potential therapeutic targets.