Background: The clinical efficacy of 1) myeloablative BuFlu and 2) PTCy as GVHD prophylaxis have been independently shown in multiple single-center studies. Here, we sought to combine these two promising strategies in the context of a multi-institutional clinical trial. Methods: IV Bu was given in pharmacokinetically adjusted doses to achieve a targeted steady-state concentration. PTCy at 50mg/kg on Days +3 and +4 was administered as sole GVHD prophylaxis. Ninety-two patients (median age 49, range 21-65) receiving HLA-matched allografts (49% related and 51% unrelated) were enrolled at three institutions: 42 at Johns Hopkins, 38 at Fred Hutchinson, and 12 at MD Anderson. Diagnoses included 37 patients with de novo AML (40%), 16 with secondary AML (17%), 15 with acute lymphoblastic leukemia (16%), 13 with myelodysplastic syndrome (14%), 5 with chronic myelogenous leukemia (5%), two with chronic myelomonocytic leukemia (2%), three with non-Hodgkin lymphoma (3%), and one with multiple myeloma (1%). Twenty-five patients (27%) were not in remission by morphologic criteria at the time of allogeneic transplantation and an additional 17 patients (18%) had flow cytometric, cytogenetic, or molecular evidence of disease. Results: Five patients (5.4%) had primary graft failure. The cumulative incidences of grades 2-4 and 3-4 acute GVHD were 51% and 16%, respectively, and the cumulative incidence of chronic GVHD was 14%. GVHD was the primary cause of death in two patients, and one patient died of veno-occlusive disease. The 100 day and 1 year non-relapse mortality were 9.8% and 16%, respectively. Relapse occurred in 24% of these high-risk patients. At 1 year, the EFS was 64% and the OS was 72%. For the 50 patients in complete remission without MRD, at 1 year the EFS was 77% and the OS was 78%. Conclusions: This multi-institutional trial confirms the efficacy of high-dose PTCy as single-agent GVHD prophylaxis and demonstrates that it can be safely and effectively combined with the myeloablative BuFlu conditioning regimen. Clinical trial information: NCT00809276.