Nestin, which was initially identified as a marker of neural stem cells, has been reported in regenerating pancreas as well as in early embryonic stem (ES) cell derivatives. However, little is known about its specific roles in stem cells as a functional regulator. We investigated the source of the action of nestin in ES and adult pancreatic ductal stem (PDS) cells in regard to the neogenesis of insulin-secreting beta-cells. In ES cells, suppression of nestin by gene silencing led to an increased expression of the pluripotency-associated genes, including Oct 4, Nanog, and SSEA-1, before embryoid body (EB) formation, whereas it reduced endodermal and pancreatic transcription factors in EBs. Inhibition of nestin expression in adult PDS cells caused a low expression of pancreatic transcription factors and islet hormones, leading to poor beta-cell development and insulin secretion. These data may indicate not only that nestin is a simple stem cell marker, but also that it constitutes a functional factor at the time of stem cell differentiation. We suggest that nestin plays pivotal roles as an intermediate regulator governing both stemness and differentiation of stem cells in the process of their differentiation into insulin-secreting cells.