AbstractHyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5B^N642H, a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5B^N642H in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5B^N642H-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5B^N642H patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5B^N642H crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5B^N642H can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5B^N642H, conferring kinetic stability to the mutant anti-parallel dimer. This study provides a molecular explanation for the STAT5B^N642H activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.