Blastocyst formation during mammalian preimplantation development is a unique developmental process that involves lineage segregation between the inner cell mass and the trophectoderm. To elucidate the molecular mechanisms underlying blastocyst formation, we have functionally screened a subset of preimplantation embryo-associated transcripts by using small interfering RNA (siRNA) and identified Bysl (bystin-like) as an essential gene for this process. The development of embryos injected with Bysl siRNA was arrested just prior to blastocyst formation, resulting in a defect in trophectoderm differentiation. Silencing of Bysl by using an episomal short hairpin RNA expression vector inhibited proliferation of embryonic stem cells. Exogenously expressed Bysl tagged with a fluorescent protein was concentrated in the nucleolus with a diffuse nucleoplasmic distribution. Furthermore, the loss of Bysl function by using RNA interference or dominant negative mutants caused defects in 40S ribosomal subunit biogenesis. These findings provide evidence for a crucial role of Bysl as an integral factor for ribosome biogenesis and suggest a critical dependence of blastocyst formation on active translation machinery.Blastocyst formation is the first differentiation process during mammalian embryonic development, leading to the segregation of two distinct cell lineages: the inner cell mass (ICM) and the trophectoderm (TE) (27). Prior to blastocyst formation, intercellular junctions are established during compaction and cellular polarity is developed. The subsequent rounds of asymmetric divisions give rise to spatially segregated outer and inner cells. The outer layer of cells adopts a polarized epithelial structure differentiating into the TE, which then contributes to extraembryonic tissues, including the placenta. The apolar ICM is the founder cell population of the embryo proper. The ICM and its successor, the epiblast, comprise transient pluripotent stem cell populations. After implantation, the cells begin to differentiate into specific cell types that will form the three primary germ layers. Embryonic stem (ES) cells, which are derived from the ICM or epiblast, self-renew and maintain pluripotent differentiation capacity in culture. Although the regulatory networks that are responsible for the maintenance of ES cell identity have been extensively investigated, molecular analysis of preimplantation development is quite limited due to the scarcity of materials and experimental approaches.With the advent of the genome projects, global gene expression profiles of various tissues and developmental stages have been generated from cDNA libraries and microarray analyses. These studies have provided an effective way to identify genes that are preferentially expressed in specific cell types or tissues, especially in early embryos from which only small amounts of material can be obtained. In addition, gene silencing by RNA interference (RNAi) has been established as a method for reverse genetic analysis of gene function in mammalian cell...