To better understand whether microRNAs (miRNAs) are involved in the self-renewal of stem cells and fate determination of neural stem cells and to identify the miRNA expression patterns of different neural stem cells (NSC) in vitro and in vivo, we examined miRNA expression profiles of murine embryonic stem cells (ESC), NSC induced from ESC and isolated from E8.5 mouse neural tube (E8.5-NSC) using microarray technique. It was found that a total of 40 miRNAs had similar expression level in all the three cells [false discovery rate (FDR)=0, fold change <3.0]. Moreover, q-PCR showed that some members of miR-106b and miR-17-92 families were expressed in the ESC, NSC induced from ESC (ESC-NSC) and hematopoietic stem cells (HSC). Bioinformatical analysis showed that 'stemness genes' (p21/CDKN1A, p57/CDKN1C and PTEN) were putative targets of miR-106b and miR-17-92 families. A total of 95 miRNAs were found to experience significant change (FDR=0, fold change >5.0) when the ESC differentiated into NSC. On the basis of miRNA, mRNA expression variance and predicted target genes of miRNA, we formulated a bioinformatical model for miRNA control of ESC-NSC differentiation. Then, the miRNA expression pattern was compared between NSC obtained in vitro and in vivo, and it was found that only 8% of miRNAs were different between the two NSCs. This study suggested that miR-106b and miR-17-92 families may promote the renewal of stem cells by targeting PTEN, p21/CDKN1A and p57/CDKN1C. Some miRNAs may play a key role in gene re-programming during ESC-NSC differentiation, and a substantial homogeneity exists between NSCs derived in vitro and those in vivo.