It should be noted that in this study, we succeeded in generating iCSCs from SW480 colon cancer cells, which have a pathogenic genome of “colon cancer”. In the current study, we were able to collect the cells with induced CSC properties based on their difference in the dye-efflux activity. We originally focused on the differences in the degree of efflux activity, and succeeded in establishing a new method to distinguish V50-cells from V0-cells in the OSK-SW480 cells by changing the concentration of VM. By using this method, we clarified that the forced expression of OSK induced not only an increase in the frequency of V0-cells existing in the SW480 cultures, but also the emergence of V50-cells that gained more enhanced effluxing activity. The efflux pump activity is an important property in CSCs, because the efflux pump eliminates metabolic products and toxic compounds. Therefore, V50-cells are considered to be better able to preserve their survival even in a hostile environment, such as following treatment with chemotherapy or metastatic regions, in comparison to V0-cells that enriched primary CSCs. CSCs are not uniform, thus it is important to consider not only the frequency of CSCs, but also the differences in the degree of their stemness. The present results indicated that the OSK-V50 cells have colonic differentiation potency in vitro and in vivo. In the immunohistological study, the tumors derived from OSK-V50 cells mimicked bona fide colon cancer tissue and keep their lineage as colon cancer. In contrast, the tumors of M-SW480 cells did not. We LDN-193189 confirmed that these phenotypes of our iCSCs were reproducible in serial transplantation experiments using xenograft models. In addition, CK20 is well known as a marker of differentiation in colorectal cancer, therefore, the expression of CK20 in the xenografts of OSK-V50 cells suggests that OSK induced the ability of the OSK-V50 cells to differentiate, leading to higher cell diversity in vivo. These findings were consistent with the principle of a hierarchy as advocated by the CSC concept. In terms of the clinical applications of these cells, such as the development of anti-colon CSC drugs, it is critical to develop tumors that recapitulate bona fide colon cancers. The previous reports did not focus on the histology of tumors derived from iCSCs in detail, such as the structure, phenotypic diversity and the lineage of the original tissues. There exist “cancers of unknown origin”, but not “cancers of no origin”. Therefore, it is a significant point that the current method can allow for tumors similar to actual human colon cancer to be formed even in the subcutaneous region in mice. In the current study, we could induce CSC properties in colon cancer cells by using an artificial system involving the forced expression of OCT3/4, SOX2 and KLF4. Although these factors were individually reported to be correlated with the malignant behavior and poor prognosis in various cancers, it is unclear whether there are any cells that spontaneously overexpress OCT3/4, SOX2 and KLF4 in colon cancer tissues.