Recent evidence supports the hypothesis that cancer stem cells are responsible for tumour initiation and formation. Using flow cytometry, we isolated a population of CD44+CD24(-) prostate cells that display stem cell characteristics as well as gene expression patterns that predict overall survival in prostate cancer patients. CD44+CD24(-) cells form colonies in soft agar and form tumours in NOD/SCID mice when as few as 100 cells are injected. Furthermore, CD44+CD24(-) cells express genes known to be important in stem cell maintenance, such as BMI-1 and Oct-3/4. Moreover, we can maintain CD44+CD24(-) prostate stem-like cells as nonadherent spheres in serum-replacement media without substantially shifting gene expression. Addition of serum results in adherence to plastic and shifts gene expression patterns to resemble the differentiated parental cells. Thus, we propose that CD44+CD24(-) prostate cells are stem-like cells responsible for tumour initiation and we provide a genomic definition of these cells and the differentiated cells they give rise to. Furthermore, gene expression patterns of CD44+CD24(-) cells have a genomic signature that is predictive of poor patient prognosis. Therefore, CD44+CD24(-) LNCaP prostate cells offer an attractive model system to both explore the biology important to the maintenance and differentiation of prostate cancer stem cells as well as to develop the therapeutics, as the gene expression pattern in these cells is consistent with poor survival in prostate cancer patients.