Putative cancer stem cells are a subpopulation of cancer cells that give rise to chemotherapy resistance and are therefore of prognostic and therapeutic interest, though their identification remains elusive in colon cancer due to lack of reliable and accurate markers. We previously identified a p53-dependent putative cancer stem cell population, the calcein low population (C loP), based on their exclusive efflux of the fluorescent dye Calcein. This functional identification method enables comparative live cell studies of subpopulations without differential toxicity that occurs with traditional Hoechst methods, which has confounded conclusions and limited the utility of this cancer stem cell marker. In this study, we examined the cancer stem cell-like properties of the C loP population in vivo in comparison with the parental and calcein-high population (C hiP) in human colon cancer xenografts. Serial dilution xenograft experiments in NOD/SCID mice revealed that the C loP is only marginally more tumorigenic compared to the C hiP or parental cells. However, serial passage of these tumors revealed that the C loP is uniquely enriched for self-renewal capacity in vivo compared to the other populations. Immunohistochemical analysis of these tumors revealed that the C loP possesses increased levels of nuclear β-catenin and furthermore, siRNA-mediated knockdown of β-catenin significantly reduced the C loP population. These findings highlight the C loP as an important subpopulation of tumor cells that are exclusively endowed with the ability to self-renew and propagate tumors. The dependency of the C loP on β-catenin provides a molecular explanation for this ability and suggests that this population can and should be therapeutically targeted by inhibition of Wnt signaling.