Allogeneic stem cell transplantation (allo-SCT) is a well-established treatment modality for children with severe aplastic anemia (SAA). Treatment failures are rare and mostly caused by graft rejection. Increasing mixed chimerism represents a stage at the very beginning of graft rejection, where immunological intervention might be an effective prophylactic approach. To substantiate this, we: (1) monitored peripheral blood cells from children with SAA after allo-SCT and performed pre-emptive immunotherapy in patients with increasing MC. In all, 23/34 courses of 32 children with SAA after allo-SCT showed a complete chimerism (CC) throughout and 10/34 developed different types of mixed chimerism (MC). Altogether, 4/10 with MC spontaneously developed decreasing MC, 2/10 courses persisted with low proportions of autologous cells below 30% (stable-MC), 4/10 developed increasing MC and one patient showed an autologous recovery. All patients with CC, decreasing MC or stable MC remained in continuous complete remission (CCR). In all, 2/4 patients with increasing MC developed graft rejection. Based on these observations, 2/4 new patients with increasing MC received low-dose DLIs prophylactically, and remained in CCR without any GVHD. These results substantiate that low-dose DLI in children with SAA and increasing MC can prevent graft rejection with a calculable risk to induce severe GVHD.