The 14-3-3 proteins are intracellular dimeric phosphoserine/threonine binding molecules that participate in signal transduction, checkpoint control, nutrient sensing, and cell survival pathways. Previous work established that 14-3-3 proteins are required in early Xenopus laevis development by modulating fibroblast growth factor signaling. Although this general requirement for 14-3-3 proteins in Xenopus early embryogenesis is established, there is no information about the specific role of individual 14-3-3 genes. Botanical studies previously demonstrated functional specificity among 14-3-3 genes during plant development. In this study, an antisense morpholino oligo microinjection approach was used to characterize the requirement for six specific 14-3-3 family members in Xenopus embryogenesis. Microinjection experiments followed by Western blot analysis showed that morpholinos reduced specific 14-3-3 protein levels. Embryos lacking specific 14-3-3 isoforms displayed unique phenotypic defects. In particular, reduction of 14-3-3 tau (τ) protein, and to a lesser extent, 14-3-3 epsilon (ϵ), resulted in embryos with prominent gastrulation and axial patterning defects and reduced mesodermal marker gene expression. In contrast, reduction of 14-3-3 zeta (ζ) protein caused no obvious phenotypic abnormalities. Reduction of 14-3-3 gamma (γ) protein resulted in eye defects without gastrulation abnormalities. Therefore, individual 14-3-3 genes have separable functions in vertebrate embryonic development. Developmental Dynamics 235:1761–1776, 2006. © 2006 Wiley-Liss, Inc.