Acute Myelogenous Leukemia (AML) is an aggressive cancer that strikes both adults and children and is frequently resistant to therapy. Thus, identifying signals needed for AML propagation is a critical step toward developing new approaches for treating this disease. Here, we show that Tetraspanin 3 is a target of the RNA binding protein Musashi 2, which plays a key role in AML. We generated Tspan3 knockout mice that were born without overt defects. However, Tspan3 deletion impaired leukemia stem cell self-renewal and disease propagation and markedly improved survival in mouse models of AML. Additionally, Tspan3 inhibition blocked growth of AML patient samples, suggesting that Tspan3 is also important in human disease. As part of the mechanism, we show that Tspan3 deficiency disabled responses to CXCL12/SDF-1 and led to defects in AML localization within the niche. These identify Tspan3 as an important regulator of aggressive leukemias and highlight a role for Tspan3 in oncogenesis. Display omitted •Expression analysis implicates Tetraspanin 3 (Tspan3) in leukemia•Tspan3 deletion impairs AML propagation and improves survival in mouse disease models•The chemokine response of AML cancer cells is impaired by Tspan3 deletion•Tspan3 knockdown impairs human AML growth in xenografts Reya and colleagues identify Tetraspanin 3 as a key signal required for AML. Tspan3 deletion leads to improved survival in mouse models of AML and reduced cancer growth in xenografts. Tspan3 loss impairs migration of leukemic cells to SDF suggesting that it may influence oncogenesis by controlling a normal chemokine response.