Objective To establish six kinds of stable embryonic stem cell culture system. Methods Mouse embryonic stem cells (ES D3 line) are maintained in following culture systems The primary mouse embryonal fibroblasts (MEF) in serum medium and MEF in serum-free medium as well as SNL (mouse fibrob last subline ) in serum medium all together consist three kinds of feeder layer dependent culture systems. The serum medium and serum-free medium supplemented w ith recombinant murine leukemia inhibitory factor (mLIF) respectively as well as Buffalo Rat Liver (BRL) conditioned medium consist three varied feeder-free lay er culture systems. The ES D3 cells are passaged 10 times in vitro and their clo ne are observed morphologically. In order to identify ES cell pluripotent and un differentiation on the above mentioned systems, we tested the alkaline phosphata se (AKP) staining which is a marker of the undifferentiated cells and the experi ment of ES D3 cells differentiation in nude mice were investigated. Results On both feeder layer systems and feeder free systems, the typical ES cells clone shape r evealed as follows nested aggregation (clone) with clear edge and smooth surfac e as well as close arrangement within the clone. Strong positive AKP reactive c ells are observed. On the other hand. the teratomas made up with various tissue s are formed in nude mice. Conclusion Each of the six culture systems can be us ed to promote the growth of ES D3 and maintain their undifferentiation and pluri potentiaty, which lay a solid foundation for the application of ES cells.