Abstract Purpose: Ewing sarcoma (ES) is considered a systemic disease with the majority of patients harboring micrometastases at diagnosis. Multiparameter flow cytometry (MPFC) was used to detect ES cells in bone marrow (BM) of ES patients at diagnosis and to evaluate the prognostic significance of CD56 expression in BM samples. Experimental Design: BM samples from 46 ES patients, 6 tumor aspirates, 2 ES cell lines, and 10 control BM samples were analyzed by MPFC. ES cells were identified by the combination of CD45−/CD90+/CD99+. CD56 was evaluated on these cells by a cutoff of 22%. Results: BM samples obtained from all patients at diagnosis were found to be positive for micrometastatic tumor cells assessed by CD99+/CD90+/CD45− expression. A total of 60% of the BM samples harbored high CD56 expression. There was a highly significant correlation between CD56 expression and progression-free survival (PFS; 69% in low/negative expression versus 30% in high expression groups, P = 0.024). In patients with localized nonpelvic disease, those expressing low/negative CD56 had 100% PFS versus 40% in the high expressing group (P = 0.02). By Cox regression analysis, CD56 was found to be an independent prognostic marker with an 11-fold increased risk for relapse in patients with localized disease (P = 0.006). Conclusion: All samples contained cells that are positive for the CD99+/CD90+/CD45− combination at diagnosis, indicating that ES is a systemic disease. CD56 expression could be used to reveal ES patients with excellent prognosis or patients predisposed to relapse, thus improving treatment stratification and implementation of personalized therapy. Clin Cancer Res; 17(9); 2900–7. ©2011 AACR.