Activation of oncogenes underlies the pathogenesis of most human cancers. In neuroblastoma, amplification of the oncogene MYCN occurs in approximately 22% of cases and is associated with advanced stages of the disease and poor prognosis. Identification of other oncogenes that are consistently mutated or overexpressed in neuroblastoma is crucial for a molecular understanding of the pathogenic process. Here, we report that the oncogene Bmi-1 is highly expressed in human neuroblastoma cell lines and primary tumors. Neuroblastoma development in MYCN transgenic mice, an animal model for the human disease, was associated with a marked increase in the levels of Bmi-1 expression. Bmi-1 cooperated with MYCN in transformation of benign S-type neuroblastoma cells and avian neural crest cells by inhibiting the apoptotic activity of MYCN. Importantly, down-regulation of Bmi-1 impaired the ability of neuroblastoma cells to grow in soft agar and induce tumors in immunodeficient mice. Moreover, Bmi-1-knockdown neuroblastoma xenografts were characterized by a significant increase in the amount of Schwannian stroma, a histological feature associated with clinically favorable neuroblastomas. These findings suggest a crucial role for Bmi-1 in neuroblastoma pathogenesis and provide insights into the molecular basis of neuroblastoma heterogeneity.