Mouse embryonic stem (ES) cells were cultured on artificial polymeric biomembranes with a phospholipid polymer (phosphatidylcholine, PC) surface. ES cells aggregated to form an embryoid body (EB) on the PC surface immediately after seeding. Single EBs formed on the PC surface after 3 d, and their size was depended on the initial number of cells that were seeded. In contrast, many small EBs with a nonuniform shape formed on a conventional hydrophobic nontreated polystyrene surface. RT-PCR assays of the Ells indicated that cell-cell interactions were enhanced in EBs that formed on the PC surface compared with EBs that formed on the polystyrene surface. The transcription factor Pax6, which is a marker of the differentiation of ES cells to neurons, was not expressed in EBs that formed on the PC surface; however, EBs that formed on the polystyrene surface did express Pax6, indicating that they were undergoing differentiation into neurons. When stimulated with retinoic acid (an inducer of differentiation into neurons), EBs on the PC surface expressed Pax6. We also observed that the adhesion of ES cells to the PC surface was reduced. Thus, the formation of large EBs on the PC surface was due to enhanced cell-cell interaction and inhibition of nonspecific differentiation to neurons.