Mouse embryonic stem (ES) cell lines, which were established by culturing on feeder cells, have usually been cultured without feeder cells in the presence of leukemia inhibitory factor. However, proliferating rate of ES cells in the condition is often lower than that with feeder cells. Here, we found that conditioned medium (CM) from feeder cells (STO cells) increased the number of undifferentiated cells in a culture dish by promoting attachment of ES cells. The attached cells were increased in 4 h after replating ES cells in the presence of CM from STO cells, and they formed flat colonies composed of undifferentiated cells. This culture system with CM from feeder cells is useful in preparing a large number of well-defined ES cells. In addition, from present experiments we found that the dish double-coated with gelatin and CM was extremely useful for culturing ES cells.