Human neuroblastoma I-type cells have been proposed as a population of malignant neural crest stem cells, based on their high tumorigenic potential, expression of stem cell markers, and ability to differentiate into cells of neural crest lineages, including neuroblastic (N-type) and Schwann/glial (S-type) cells. Here, we demonstrate at single cell levels that a subpopulation of I-type cells possess clonogenic self-renewal capacity that requires the Polycomb group family transcription repressor Bmi-1. We further show that Bmi-1 expression levels exert an instructive influence on lineage commitment by I-type cells. Spontaneous and induced differentiation of I-type cells into S-type cells is accompanied by a marked reduction in the level of Bmi-1 expression, and enforced down-regulation of BMI-1 facilitates spontaneous differentiation of I-type cells into S-type cells. By contrast, N-type neuroblastoma cell lines and differentiated N-type cells express higher levels of Bmi-1 relative to I-type cells, and overexpression of BMI-1 promotes the differentiation of I-type cells along the neuronal lineage. Thus, Bmi-1 acts in a concentration-dependent manner in the control of the delicate balance between the self-renewal and differentiation of neuroblastoma I-type cells. These observations suggest that graded activation of a master regulator within individual tumors could trigger divergent developmental programs responsible for both tumor growth and heterogeneity. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.