Xenograft models are invaluable tools in establishing the current paradigms of hematopoiesis and leukemogenesis. The zebrafish has emerged as a robust alternative xenograft model but, like mice, lacks specific cytokines that mimic the microenvironment found in human patients. To address this critical gap, we generated the first “humanized” zebrafish that expresses human hematopoietic-specific cytokines (granulocyte-monocyte colony-stimulating factor, stem cell factor, and stromal cell-derived factor 1α). Termed GSS fish, these zebrafish promote survival, self-renewal and multilineage differentiation of human hematopoietic stem and progenitor cells and result in enhanced proliferation and hematopoietic niche-specific homing of primary human leukemia cells. Using error-corrected RNA sequencing, we determined that patient-derived leukemias transplanted into GSS zebrafish exhibit broader clonal representation compared to transplants into control hosts. GSS zebrafish incorporating error-corrected RNA sequencing establish a new standard for zebrafish xenotransplantation which more accurately recapitulates the human context, providing a more representative cost-effective preclinical model system for evaluating personalized response-based treatment in leukemia and therapies to expand human hematopoietic stem and progenitor cells in the transplant setting.