ES cells can provide a useful system in vitro for studing differentiation and development in the absence of differentiation inhibitory factor (DIF) or leukemia inhibititory factor (LIF).The feeder layer can produce LIF which can maintain ES cells in the state of undifferentiation and development.We chosed 10 d,14 d,18 d pregnant mice to produce feeder layer.The results showed that feeder layer from 10d to 18 d pregnant mice cultured ES-D 3 all grew well.