ARTICLE ABSTRACTJSRV, a simple beta-retrovirus, is the etiologic agent of ovine pulmonary adenocarcinoma, a form of non–small cell lung cancer in sheep and goats. It has been shown that the envelope protein alone is sufficient to induce tumorigenesis in the lungs of mice when delivered via an adeno-associated viral vector. Here, we tested the hypothesis that JSRV envelope–induced tumors are maintained by a small population of tumor-initiating cells, termed cancer stem cells. To test this hypothesis, dissociated cancer cells were sorted from envelope-induced tumors in mouse lung based on the putative stem cell markers Sca-1, CD34, and CD133, the pluripotency-associated transcription factor Oct4, and the level of Wnt signaling. No association with increased tumor-initiating capacity was found with any of the cell-surface markers. In addition, we were unable to detect any evidence of Oct4 expression in tumor-bearing mouse lung. However, tumor cells possessing an active Wnt signaling pathway did show a significant correlation with increased tumor formation upon transplantation. Limiting dilution transplant analysis suggests the existence of a large fraction of cells with the ability to propagate tumor growth, with increasing tumor initiation potential correlating with activated Wnt signaling. Mol Cancer Res; 10(1); 86–95. ©2011 AACR.