MTX is a standard component of acute GVHD prophylaxis. However, its use can be limited by toxicity. On the basis of disease risk, we prospectively assigned 132 consecutive patients from January 2005 to February 2011 undergoing first allogeneic hematopoietic cell transplant after conditioning with fludarabine and melphalan to acute GVHD prophylaxis with tacrolimus/MTX (TAC/MTX, N = 22), TAC/micro-dose MTX/mycophenolate mofetil (TAC/μMTX/MMF, N = 78) or TAC/MMF (TAC/MMF, N = 32), to optimize acute GVHD prevention and decrease mortality. The median (range) follow-up was 24 (0.8–60) months. The median patient ages (range) were 37 (23–63), 56 (20–68) and 54 (22–68) years (P < 0.0001) for TAC/MTX, TAC/μMTX/MMF and TAC/MMF, respectively. The 100-day cumulative incidences of grade III–IV acute GVHD were 19, 23 and 49% (P < 0.015), respectively. The cumulative incidences of severe chronic GVHD at 1 year were 38, 29 and 79% (P < 0.001), respectively. Regimen-related toxicities were not significantly different among the three prophylaxis regimens. PFS and OS were equivalent between the TAC/MTX and TAC/μMTX/MMF arms despite significantly older patients in the latter arm, and both had superior PFS and OS than the TAC/MMF arm. Acute GVHD prophylaxis with TAC/μMTX/MMF is as effective as TAC/MTX and superior to TAC/MMF.