Abstract: Objective To solve the problem known as 2-cell block in in vitro culture of preimplantation embryos, modification of the nutritional components in potassium simplex optimized medium (KSOM) was attempted and the preimplantation development and cleavage of mouse embryos in such medium observed.Method One-cell mouse embryos collected from the oviduct of the superovulated mice were cultured in microdrops of different culture media for 5 days [144h after human chorionic gonadotrophin (hCG) injection], and the percentages of embryos developed to pre-blastocyst, unhatched blastocyst, partially hatched blastocyst and completely hatched blastocyst were recorded at deferent time points (96,120 and 144h post-hCG) and total cell number of blastocysts at 120 h post-hCG counted. With these indices as the evaluation criteria, we evaluated the modifications of the concentrations of glucose (0.2,5.56mmol/l), bovine serum albumin (BSA,1 and 4mg/ml), and amino acids (1/2X NEAA and EAA,or not) in KSOM for their effect on the development of Kunming mouse zygotes in vitro.Results Increasing the concentration of glucose and BSA was not shown to have significant effects on the rate of blastocyst formation at 120h post-hCG or the total cell counts in KSOM without amino acids. After supplementation of the medium with amino acids, the rate of partially and completely hatched blastocyts was significantly increased in KSOM or KSOM supplemented with high concentrations of glucose and BSA (KSOMGB). Glucose at the high concentration of 5.56mmol/L did not inhibit the development of mouse zygotes to hatched blastocysts cultured in KSOM supplemented with amino acids.Conclusion Increasing the concentration of glucose and BSA and amino acids in KSOM can promote the preimplantation development of mouse zygotes in in vitro culture.