Objective To compare the effects of supporting mouse blastocyst development and differentiation in sequential culture media and non-sequential culture media. Methods Zygotes were cultured in media Q-three for 48h after 48h of culture with media Q-one, while zygotes were cultured only in media Q-one in control group. Results The percentage of total blastocysts and the percentage of hatching blastocysts in sequential culture group were significantly higher than in non-sequential culture group, 89% vs. 12% and 38% vs. 0 respectively. Morever, the formation of expanding blastocyst was morphologically better in sequential media. Conclusions The sequential culture system was beneficial for mouse embryo development and differentiation from early embryo stage to blastocyst stage.