Leukemias are cancers of the blood cells that cause serious illness and death in children and adults. Even patients who are successfully cured of their disease often suffer from long-term adverse health effects of their curative treatment. Thus, there is a need for more targeted, less toxic, and more effective treatments. Our studies focus on the defects and mechanisms that induce leukemia by disrupting the normal growth controls that regulate blood-forming stem cells. Using a comparative genomics approach we identified genes that are differentially expressed in leukemia stem cells. These genes have been the focus of our studies to establish better biomarkers and treatment targets. A promising candidate identified in our comparative gene expression approach encodes a cell surface protein that is preferentially expressed on leukemia stem cells. We utilized this cell surface protein as a marker to isolate the rare population of cells in human leukemias with stem cell properties. This technical approach resulted in the isolation of leukemia stem cell populations that are more highly enriched than those obtained using previous techniques. The highly enriched sub-population of leukemia stem cells was used for comparative gene expression profiling to identify genes that are differentially expressed between highly matched populations of leukemia cells that are enriched or depleted of leukemia stem cells. Bioinformatics analysis of the dataset identified major cell cycle differences that distinguish human leukemia stem cells induced by abnormalities of the MLL oncogene. The distinctive cell cycle characteristics of the cells were confirmed in functional assays for their specific contributions to leukemia stem cell function and leukemia pathogenesis. These studies are the first to mechanistically link a cell surface protein with regulation of self-renewal, a key attribute of leukemia stem cells. Characterization of the crucial growth controllers that go awry in blood stem cells to cause leukemia suggests new drug targets for more effective and less toxic treatments against these devastating, life-threatening diseases.