Abstract The cancer stem cell (CSC) hypothesis provides a foundation for understanding cancer development and carries significant implications relevant to drug discovery. The generation of cell lines with cancer stem cell properties would be an important advancement in the field. If CSC were capable of indefinite self-renewal without immortalization, it should be possible to develop conditions that select this minority population from a tumor for continuous culture as a line. By employing defined serum-free media and conditions that are strongly selective for fetal colon epithelial progenitor cells, we have been able to select a morphologically homogeneous minority population, hereafter we termed cancer stem-like cells (CSLC) from a repertoire of primary and metatstatic colon cancer specimens. Six colon CSLC lines have been generated, some of which have been stably maintained in vitro for over 200 generations. When implanted subcutaneously, under the subrenal capsule, or orthotopically in immune deficient mice, all CLSC form tumors that are morphologically and phenotypically recapitulate the patient's original tumor. Furthermore, some of the lines form distant metastasis with consistent colorectal cancer morphology. All lines express CEA; however, only a fraction expresses elevated levels of “canonical” CSC markers, including CD44 or CD133. Affymetrix U133 micro-array analysis identified a set of transcripts that were elevated in the CSLC lines compared to a series of control cell populations. These included ALDH1A1, a well known colon CSC marker, and OLFM4, a recently identified marker of normal colon crypt stem cells. Whole-cell immunizations of mice with several lines has yielded a panel of CSLC-reactive monoclonal antibodies that recognize broadly expressed colon cancer antigens as well as surface proteins more discreetly represented within colon tumors and possibly representing CSC-specific markers. These lines and their reactive antibodies provide the basis for developing and characterizing novel approaches aimed at targeting and the elimination of the CSC population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5211. doi:10.1158/1538-7445.AM2011-5211