After fertilization, maternal factors direct development and trigger zygotic genome activation (ZGA) at the maternal-to-zygotic transition (MZT). In zebrafish, ZGA is required for gastrulation and clearance of maternal messenger RNAs, which is in part regulated by the conserved microRNA miR-430. However, the factors that activate the zygotic program in vertebrates are unknown. Here we show that Nanog, Pou5f1 (also called Oct4) and SoxB1 regulate zygotic gene activation in zebrafish. We identified several hundred genes directly activated by maternal factors, constituting the first wave of zygotic transcription. Ribosome profiling revealed that nanog, sox19b and pou5f1 are the most highly translated transcription factors pre-MZT. Combined loss of these factors resulted in developmental arrest before gastrulation and a failure to activate >75% of zygotic genes, including miR-430. Our results demonstrate that maternal Nanog, Pou5f1 and SoxB1 are required to initiate the zygotic developmental program and induce clearance of the maternal program by activating miR-430 expression.