Background: Novel in vitro methods surpassing limitations of current gastrointestinal cancer models such as gastric and esophagus cancer are required to functionally validate putative oncogenic loci discovered by genome sequencing efforts. The in vitro culture of primary, non-transformed tissues as three-dimensional organoids that accurately recapitulate organ structure and physiology has diverse applications including cancer biology. Methods: Mouse wild type, or p53^flox/floxin tandem with lox-stop-lox KRAS^G12D upper digestive tract tissue containing epithelial and mesenchymal components were cultured in an air-liquid-interface and subjected to adenovirus expressing either immunoglobulin Fc (control) or GFP tagged Cre recombinase. Results: 3-dimensional organoids were generated with histological adherence to normal tissue architecture including that seen in esophagus and were able to be maintained in long term culture. Organoids exposed to GFP tagged Cre adenovirus demonstrated green fluorescence not seen in organoids exposed to control virus. Conditional allele organoids that were exposed to Cre adenovirus demonstrated increased rate of growth compared to controls. Histology of these rapidly growing organoids demonstrated cellular features consistent with dysplasia. Conclusions: 3-dimensional organoids can be generated from upper digestive tract tissues, can undergo adenoviral mediated transfection to achieve oncogenic gene expression or inactivation resulting in dysplastic morphology. 3-dimensional organoids are therefore an attractive model to study or identify candidate oncogenic loci identified by recent genomic sequencing studies.