In order to maintain embryonic stem cells (ES cells) in a proliferative, unidifferentiated state in vitro. Mitomycin C-treated primary mouse embryo fibroblasts (PMEFs) were used as the feeder cells in media of DMEM supplement with LIF for co-culture with ES-D3 cells. ES cell colonies grow in small clusters. Many laboratories including ours, find that culture on feeder layers of PMEFs yields high quality ES cells with greater consistency. Some techniques that maintaining ES cell in a pluripotent state were be discussed in detail.