In embryos of ascidians, which are invertebrate chordates, the expression of a small number of genes from the zygotic genome begins between the 8- and 16-cell stages. They are considered the first zygotic genes to be expressed under the direct control of maternal factors. The initial transcriptional quiescence before the 8-cell stage is essential for establishing differential gene expression patterns between the animal and vegetal hemispheres, because these hemispheres are first segregated into distinct blastomeres at the 8-cell stage. While the mechanisms of this transcriptional silencing have not been understood, the mechanism by which maternal factors cooperatively establish the first differential gene expression patterns has been well understood. Here, we review the initial processes that occur until the 16-cell stage in ascidian embryos.