The recently developed system to culture human embryos in vitro through the implantation period has provided a unique opportunity to obtain human early trophoblast cells that were previously inaccessible by other means. The objective of this study is to identify the presence and localization of several novel early trophoblast markers that have only been described in a trophoblast cell model derived from embryonic stem cells (ESC) by BMP4 exposure. Research study Vitrified day (ED) 5 human blastocysts were warmed using the Kitazato protocol, and blastocysts recovered for two hours at 37°C in IVC1 (Cell Guidance Systems) before they were dezonated using Acid Tyrode’s solution. Embryos were then plated onto optical-grade outgrowth plates coated with fibronectin. Embryos were cultured in IVC1 for 48 h when attachment was assessed and medium exchanged with IVC2 (Cell Guidance Systems). Media was exchanged daily until fixation on ED 10 or 11. Embryos were then fixed and selectively stained with antibodies against F-actin, POU5F1, novel trophoblast markers VTCN1, WFDC2, ACTC1 and GABARP, as well as known trophoblast markers CGB, KRT7 and GATA3. Cell nuclei were identified by DAPI staining. Images were obtained by 3D confocal microscopy and analyzed by Image J software. The trophoblast cell lineage in outgrowth embryos was confirmed by positive KRT7 and GATA3 staining. Syncytiotrophoblast was further confirmed by the formation of multinucleated structures and positive CGB staining on the periphery of the colony. Novel trophoblast marker VTCN1 showed a homogenous cytoplasmic distribution throughout the trophoblast area, while WFDC1 also demonstrated cytoplasmic distribution in the same area but was more abundant toward the peripheral syncytium. GABARP demonstrated peculiar filamentous formation and strong positive staining close to the epiblast cells, which were POU5F1 positive. ACTC1 also had strong positive staining co-localized with nuclei throughout the entire trophoblast area. We have identified the expression and localization of four trophoblast markers in implantation stage human embryos in extended outgrowth culture that were previously reported in trophoblast cells differentiated from human ESC by BMP4 exposure. Importantly, this work demonstrates the potential of this newly developed extended in vitro culture system to characterize and study early trophoblast cell differentiation and examine the earliest events of pregnancy in humans that has previously been inaccessible to investigators.