Elevated cell death in human preimplantation embryos is one of the cellular events compromising pregnancy rates after assisted reproductive technology treatments. We therefore explored the molecular pathways regulating cell death at the blastocyst stage in human embryos cultured in vitro. Owing to limited availability of human embryos, these pathways were further characterized in mouse blastocysts. Gene expression studies revealed a positive correlation between the cell death index and the expression of Bcl-x transcript. Cell death activation in human blastocysts was accompanied by changes in Bcl-x splicing, favoring production of Bcl-xS, an activator of cell death. Expression of Bcl-xS was detected in a subset of human blastocysts that show particular clustering in dying and/or dead cells. Altering the Bcl-xL/Bcl-xS ratio in mouse embryos, in antisense experiments, confirmed that upregulation of Bcl-xS, with concomitant downregulation of Bcl-xL, compromised developmental potential and committed a subset of cells to undergoing cell death. This was accompanied by increased accumulation of reactive oxygen species levels without any impact on mtDNA content. In addition, altered Bcl-x splicing in favor of Bcl-xS was stimulated by culture in HTF medium or by addition of excessive glucose, leading to compromised embryo development. Thus, we conclude that inappropriate culture conditions affect Bcl-x isoform expression, contributing to compromised preimplantation embryo development.