Barrett's esophagus (BE) is the replacement of the normal esophageal squamous epithelium by a columnar lining epithelium. It is a premalignant condition for the development of adenocarcinoma of the esophagus and esophagogastric junction. BE is associated with gastroesophageal reflux which might change the expression profile of key transcription factors involved in the establishment of tissue differentiation, namely, SOX2 (associated with esophageal and gastric differentiation) and CDX2 (associated with intestinal differentiation). Here, we sought to characterize the expression profile of SOX2 and CDX2 in the sequential alterations of the esophageal mucosa towards adenocarcinoma and compare it with the well-established gastric and intestinal mucin profiles (MUC5AC, MUC6, and MUC2). We observed that SOX2 and CDX2 expression correlates with gastric and intestinal differentiation in BE, defined by morphological parameters and mucin expression. We show the presence of a complete intestinal profile in BE, without gastric mucins and without SOX2, and we observed an evolutionary modulation of the metaplastic phenotype by SOX2 and CDX2. We observed that adenocarcinomas harbor more frequently a mixed gastric and intestinal phenotype. In conclusion, our study establishes a role for transcription factors SOX2 and CDX2 in the progression from gastric to gastrointestinal differentiation in Barrett's metaplasia.