Ewing sarcoma is the second most frequent primary tumor of bone, preferentially occurring in children and adolescents. Despite significant improvements have been achieved in localized tumors thanks to dose intensification of chemotherapy, outcome of patients with metastasis at diagnosis remains grim. In addition, even in the most favorable situations, patients must face with important side effects which significantly impact their quality of life. Either scientific and patient communities are now very sensitive on the need of new drugs, which may reduce chemotherapy toxicity while maintaining effectiveness of current regimens. We present here a new engeneered human bivalent single chain fragment variable diabody (C7 scFv diabody) directed against CD99, a transmembrane protein whose high expression characterizes Ewing sarcoma. The triggering of CD99 with C7scFv diabody induces rapid and massive Ewing sarcoma cell death through MDM2 ubiquitination and p53 reactivation. Accordingly, the most CD99-responsive Ewing sarcoma cells have transcriptional active p53 and greatly benefit from MDM2 degradation. CD99 triggering also potentiates the cytotoxic effect of doxorubicin in vitro and in vivo and reactivates p53 to a much greater degree, which in turn markedly increased expression of pro-apoptosis genes. Evalua- tion of Ki-67 labelling and apoptosis rate by TUNEL confirms the efficacy of the treatment in xenografts. In contrast, mesenchymal stem cells, though expres- sing high levels of CD99, show no p53 activation and escape death induced by CD99 C7scFv diabody. No sign of toxicity was observed in mice treated with anti-CD99 scFV C7 diabody. Overall, our data provide the rationale for the use of this newly developed anti-CD99 diabody in the treatment of patients with Ewing sarcoma.