In recent years, applications of stem cells have already involved in all domains of life science and biomedicine. People try to establish human embryonic stem cell lines (hESCs) in order to carry out hESC-related studies. In this study, we explored what embryos are conducive to the establishment of hESCs. The discarded embryos from in vitro fertilization–embryo transfer (IVF–ET) cycles were sequentially incubated into blastocysts, and then the inner cell mass (ICM) was isolated and incubated in the mixed feeder layer. The cell lines which underwent serial passage were identified. After a total of 1,725 discarded embryos from 754 patients were incubated, 448 blastocysts were formed with 123 high-quality blastocysts. The blastulation rate was significantly higher in the discarded embryos with non-pronucleus (0PN) or 1PN than in the discarded embryos with 2PN or ≥3PN. The blastulation rate of the D3 embryos with 7–9 blastomeres was higher. Among the originally incubated 389 ICMs, 22 hESCs with normal karyotype were established, and identified to be ESCs. Therefore, in establishing hESCs with discarded embryos, D3 0PN or 1PN embryos with 7–9 blastomeres should be first selected, because they can improve high-quality blastulation rate which can increase the efficiency of hESC establishment. J. Cell. Biochem. 113: 3835–3842, 2012. © 2012 Wiley Periodicals, Inc.