The Ewing family of tumors (EFT) is an important group of pediatric malignancies with a guarded prognosis. Little is known about the heterogeneity of EFT cells, and the cellular origin of EFT is disputed. We now add evidence that EFT are heterogeneous by showing that EFT cells from spheres growing in serum-free medium are markedly more tumorigenic than adherently growing EFT cells. Furthermore, EFT cells strongly expressing CD57 (HNK-1), a surface marker for migrating and proliferating neural crest cells, are more tumorigenic than cells with low expression of CD57, possibly mediated in part by enhanced adhesion and invasion. We contribute to the controversy about the cellular origin of EFT by clonal analysis, showing that EFT cells can differentiate similar to neural crest cells. These data increase our knowledge about the pathogenesis and heterogeneity of EFT.