This article describes an in vitro culture system for embryonic stem (ES) cells, which are expected to serve as a cell source for transplantation because of their potential for indefinite expansion and pluripotency. We present a serial passaging protocol that permits the enrichment of undifferentiated ES cells by culturing them on a surface modified with a synthesized dendrimer having d-glucose as a functional ligand. The d-glucose-displaying dendrimer (GLU/D) surface caused mouse ES cells to form loosely attached spherical colonies, and the frequency of such colonies increased gradually with the number of passages. Analyses of alkaline phosphatase activity and the gene expression of pluripotency and early differentiation markers revealed that the spherical colony cells passaged four times (a total of 16days in culture) on the GLU/D surface acquired more of the characteristics of undifferentiated cells than the cells cultured on a conventional gelatin-coated surface. Moreover, the cells cultured on the GLU/D surface retained their germ-line transmission ability after four passages. These results indicate that this modified culture surface may be a useful tool for obtaining enriched preparations of undifferentiated ES cells.