Pathophysiology of graft failure occurring after allogeneic hematopoietic stem cell transplantation still remains elusive. We measured serum levels of several different cytokines/chemokines in 15 children experiencing graft failure, comparing their values with those of 15 controls who had sustained donor cell engraftment. Already at day +3 after transplantation, patients developing graft failure had serum levels of IFNγ and CXCL9, a chemokine specifically induced by IFNγ, significantly higher than those of controls (8859±7502 versus 0 pg/ml, p=0.03, and 1514.0±773 versus 233.6±50.1 pg/ml, p=0.0006, respectively). The role played by IFNγ in hematopoietic stem cell transplantation-related graft failure was further supported by the observation that a rat anti-mouse IFNγ-neutralizing monoclonal antibody promotes donor cell engraftment in Ifngr1-/- mice receiving an allograft. In comparison to controls, analysis of bone marrow-infiltrating T lymphocytes in patients experiencing graft failure documented a predominance of effector memory CD8+ cells, which showed markers of activation (over-expression of CD95 and down-regulation of CD127) and exhaustion (CD57, CD279, CD223 and CD366). Finally, we obtained successful donor engraftment in 2 out of 3 children with primary hemophagocytic lymphohistiocytosis who, after experiencing graft failure, were re-transplanted from the same HLA-haploidentical donor under the compassionate use coverage of emapalumab, an anti-IFNγ monoclonal antibody recently approved by the Food and Drug Administration for treatment of patients with primary hemophagocytic lymphohistiocytosis. Altogether, these results suggest that IFNγ pathway plays a major role in graft failure occurring after hematopoietic stem cell transplantation. Increased serum levels of IFNγ and CXCL9 represent potential biomarkers useful for early diagnosis of graft failure and provide the rationale for exploring the therapeutic/preventive role of targeted neutralization of IFNγ.