Clinically useful pre-transplant predictive factors of acute graft-versus-host-disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT) are lacking. We prospectively analyzed HSC graft content in CD34+, NK, conventional T, regulatory T and invariant natural killer T (iNKT) cells in 117 adult patients before allo-SCT. Results were correlated with occurrence of aGVHD and relapse. In univariate analysis, iNKT cells were the only graft cell populations associated with occurrence of aGVHD. In multivariate analysis, CD4− iNKT/T cell frequency could predict grade II-IV aGVHD in bone marrow and peripheral blood stem cell (PBSC) grafts, while CD4− iNKT expansion capacity was predictive in PBSC grafts. Receiver operating characteristic analyses determined the CD4− iNKT expansion factor as the best predictive factor of aGVHD. Incidence of grade II-IV aGVHD was reduced in patients receiving a graft with an expansion factor above versus below 6.83 (9.7 vs 80%, P < 0.0001), while relapse incidence at two years was similar (P = 0.5).The test reached 94% sensitivity and 100% specificity in the subgroup of patients transplanted with human leukocyte antigen 10/10 PBSCs without active disease. Analysis of this CD4− iNKT expansion capacity test may represent the first diagnostic tool allowing selection of the best donor to avoid severe aGVHD with preserved graft-versus-leukemia effect after peripheral blood allo-SCT.