We compared the characteristics of the method establishing embryonic stem cell lines from five different mouse strains using the medium containing 70% rat heart cell-conditioned medium (RH-CM) as ES cell culture medium, using the primary murine embryo fibroblast as feeder cells, and using the digestive enzyme buffer containing 1% chicken serum and "the series digestive method". We first reported new ES cell lines established from the outbred strain mice KM and ICR using the improved method in our lab and the ratio of establishment of ES cell lines from KM and ICR strain mice is up to 12% and 42.1% respectively. Compared with routine method of establishing ES cell lines, the improved method made distinct differences, increasing the ratio of ES cell line's establishment of 129/ter mouse from 11.8% to 33.3%, that of C57BL/6J mouse from 3.7% to 13.3%, that of BALB/c mouse from 2.9% to 19.4%. We tested the appropriate dispersing occasion, that is proliferating period of the ICM, affected the formation of ES clones and the ratios of ES cell lines established. It was shown that the most appropriate dispersed occasion for the ICM of 129/ter, C57BL/6J, BALB/c, KM and ICR mice was 4-6 d, 3-3.5 d, 4 d, 4-5 d, 4-5 d after ICM proliferation respectively. At the same time, the effects of the concentration of digestive enzyme buffer were discussed. It was found that the ES cells from BALB/c mice were sensitive to the high concentration of digestive enzyme buffer and the 0.05% Trypsin-0.008% EDTA is an ideal concentration for their establishment and maintenance. It was shown that 'the series dispersed method' was much better than 'the once dispersed method' on the aspect of dispersing the proliferating ICM and formation of ES clones. Compared with the routine ES cell culture medium containing mLIF, the RH-CM not only remarkably inhibited the differentiation of murine ES cells and maintained their diploid karyotype, but also promoted the attachment and growth of ES cells. This improved method of establishment and culture of ES cell lines effectively maintained a series of their characteristics of pluripotent embryonic stem cells.