Despite advances in cancer treatment, relapse remains a critical factor in the overall mortality of patients. Relapse is often driven by a rare population of cells within the tumor that have the ability to evade treatment, self-renew, and repopulate a tumor. The initiating cell in leukemia is often a normal hematopoietic stem cell that accumulates mutations over time, causing aberrant function. These cells, often referred to as cancer stem cells, co-opt stem cell programs. These cancer stem cells maintain their ability to interact with and manipulate the stem cell niches within the bone marrow microenvironment to further propagate disease. To understand the programs that are necessary for successful leukemia engraftment and progression, the Reya lab conducted an in vivo CRISPR screen. From the dropouts of this screen, we targeted GFER. GFER, a secreted protein, could potentially influence the surrounding niche to make it more permissive to the cancer stem cell. We found that the inhibition of GFER led to mislocalization within the bone marrow as well as a decrease in leukemia blasts in the bone marrow and spleen. This could be evidence that the cancer stem cell relies on the niche and, without it, they are left vulnerable. To further target the cancer stem cell and its relationship with the niche, we developed a monoclonal blocking antibody that binds to Tspan3. This anti-Tspan3 antibody reduced colony forming ability of myeloid leukemia patient samples, both in vitro and in vivo. We found that inhibition of Tspan3, by either blocking antibody or shRNA, led to alterations in stem programs that could push the cancer stem cell to differentiate. This would leave the cancer stem cell more susceptible to traditional cancer treatments. Indeed, we found that the anti-Tspan3 antibody synergized with imatinib, a front-line tyrosine kinase inhibitor for the treatment of CML. To further develop the antibody, we used humanized variants to test against myeloid leukemia cells. These humanized constructs similarly inhibited colony formation and they would be less immunogenic when delivered to patients in the future. The studies contained here illustrate a basis for targeting the interface between cancer stem cells and their microenvironment for the development of novel therapies.