Embryonic stem (ES) cells can differentiate into many cell types. Recent reports have shown that ES cells can differentiate into insulin-producing cells. We have established an ES cell line in which exogenous Pdx-1 expression was precisely regulated by the Tet-off system integrated into the ROSA26 locus and succeeded to produce insulin-producing cells. The Pdx-1 expressing final differentiated insulin-positive cells can be maintained for more than 2 months. However, in spite of their induced expression of Pdx1, the repeated passages of cells lost their capacity to express insulin and NeuroD1 gene. Forced expression of NeuroD1 gene by adenoviral vector in these cells restored the expression of insulin. These results suggested that maintenance of the property of insulin-producing cells defived from ES cells could be achieved by synergistic expression of Pdx-1 and NeuroD1. (c) 2007 Elsevier Ireland Ltd. All rights reserved.