Diminished successes of chemotherapy treatment, recurrent disease and tumor resistance to anticancer therapy have all been attributed to intra-tumoral differences among sub-population of cancer cells, and to the existence of cancer stem cells with the capacity for self-renewal. In our laboratory, it has been shown that the use of an experimental platform comprising human embryonic stem cell (hESC)-derived cellular microenvironment in immune-compromised mice enables functional distinction of heterogeneous tumor cells; including cells that do not grow into a tumor in the conventional platform of a direct tumor xenograft. We used six clonally expanded sub-populations derived from ovarian clear cell carcinoma of a single tumor, to demonstrate striking intra-tumoral heterogeneity that has a component which is inherent to subpopulation and hence durable and a component which is dynamically dependent on the tumor microenvironment. Each of six clonally expanded sub-populations displays a different level of both morphologic and tumorigenic differentiation, and properties of self-renewal, often attributed to cancer stem cells. We have shown that the human embryonic stem cell (hESC)-derived cellular microenvironment exposes the functional distinction of heterogeneous cancer stem cell ?sub-populations ? and learned that ovarian cancer cells display microenvironment-dependent plasticity with the ability to differentiate and then restore the capacity for self-renewal ? . We delineate the distinct gene expression profile and epigenetic landscape of two such sub-populations representing the extremes of phenotypic heterogeneity in terms of niche dependent-self-renewal and tumorigenic differentiation and present a suite of robust differences in the genes and pathways that underlie this intra-tumoral phenotypic heterogeneity. Importantly, we demonstrated that a single ovarian tumor may contain multiple sub-populations of cancer cell which exhibits major differences in theirs sensitivity to different drug compounds. This research presents a view into the vast extent of cancer cell heterogeneity in solid tumors and indicates the urgent need for the development of clinically relevant characterization procedures for the different sub-population which exist within a single tumor and for the use of multimodal anti-cancer therapeutic strategies to eliminate both the heterogeneous cancer stem cells and their differentiated derivatives.