We have recently demonstrated that human paediatric mesenchymal stem cells can be reprogrammed toward a Ewing’s sarcoma family tumor (ESFT) cancer stem cell (CSC) phenotype by mechanisms that implicate microRNAs (miRNAs). Here, we show that the miRNA profile of ESFT CSC is shared by embryonic stem cells and CSC from divergent tumor types. We also provide evidence that the miRNA profile of ESFT CSC is the result of reversible disruption of TARBP2-dependent miRNA maturation. Restoration of TARBP2 activity and systemic delivery of synthetic forms of either of two of its targets, miRNA-143 or miRNA-145, inhibited ESFT CSC clonogenicity and tumor growth in vivo. Our observations suggest that CSC self-renewal and tumor initiation may depend on deregulation of TARBP2-dependent miRNA expression. Overall design: 2 samples of primary Ewing sarcomas, divided into CD133+ and CD133- fractions. One sample of EWS-FLI1 expressing human pediatric mesenchymal stem cells, divided into CD133+ and CD133- fractions. One sample of STA-ET-8.2 cells, divided into CD133+ and CD133- fractions.