APC10.1 is a new intestinal cell line derived from ApcMin/+ mice that retains both the heterozygous Apc genotype and a nonactivated Wnt signaling pathway and displays an early neoplastic phenotype. Although tumorigenic both in immunodepressed and in immunocompetent syngeneic mice, it requires a high cell dose and a long latency. Its epithelial/intestinal origin is shown, in a gene expression profile, by the expression of epithelial transcripts (such as cytokeratin and laminin isoforms) and of developmental regulatory genes (such as Tcf-4, Hnf3beta, p21, Ihh, Hes1) necessary for, or involved in, the maintenance of intestinal stem cells. The lack of activation of the Wnt cascade in APC10.1 cells is shown both by the expression profile of Wnt target genes and by the standard TCF reporter assay. APC10.1 cell line is a novel in vitro model that can contribute to a better understanding of the clinical evolution of familial adenomatous polyposis and to finding of new prophylactic and therapeutic approaches. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.