Maternal periconceptional alcohol (PC-EtOH) exposure in the rat causes fetal growth restriction and sex-specific changes to placental morphology in late gestation. This may derive from perturbations to the pre-implantation embryo and/or its capacity to form a placenta. This study aimed to examine cell allocation in the pre-implantation embryo and trophectodermal (TE) derivatives after PC-EtOH exposure. Sprague Dawley dams were administered 12.5% v/v EtOH or a control diet from 4 days prior (E-4) to 4 days after conception (E4) in a liquid diet. No differences were found between control and PC-EtOH in forming a competent blastocyst at E5, assessed by total cell number and cell allocation to the TE or the inner cell mass. However, PC-EtOH embryos showed significant reductions in nuclear TE CDX2 fluorescence intensity (P<0.0001), which may represent either delayed formation of the TE or lead to precocious differentiation to the placental trophoblasts. To assess the invasive capacity of PC-EtOH embryos, a subset of in vivo derived embryos at E5 were cultured in vitro for 6 days. Embryos exposed to in vivo PC-EtOH showed reduced trophoblast outgrowth area (P<0.05), and the largest of the pan-cytokeratin positive trophoblasts; the parietal trophoblast giant cells (P=0.01). This study shows in vivo PC-EtOH can affect differentiation and invasive capacity of the TE lineage, which may contribute to altered placental development, fetal growth restriction and the programming of adult disease.