In early mouse embryos, gastrulation forms the three germ layers from which all cells of the adult derive. The signalling factors and gene regulatory networks (GRN) involved in the formation of the human and mouse endoderm have not been fully elucidated. We have focused on Mixl1, which is expressed in the primitive streak during gastrulation and is vital for the formation of endoderm. Previous in vitro studies have shown that Mixl1 expression is necessary for both mesodermal and endodermal differentiation. Furthermore, a previous study within the lab found a correlation between the timing of Mixl1 expression and germ layer specification. However, the role of Mixl1 expression timing as well as the direct targets of Mixl1 had not been fully explored. In determining how timed Mixl1 expression could be responsible for both mesoderm and endoderm differentiation, we used mouse epiblast stem cells (EpiSC) that are inducible for Mixl1. Using timed doxycycline inductions of Mixl1, we have performed the first Mixl1 ChIP-seq analysis in a proper mouse gastrulation model and have built a comprehensive GRN centred on Mixl1 by combining ATAC-seq and RNA-seq in the same cell conditions and reanalysing published data on promoter capture Hi-C in EpiSC. The early induction of Mixl1 during gastrulation results in the inhibition of T/Brachyury and the expression of the anterior primitive streak (APS) markers, Gsc and Lhx1, from where the endoderm progenitors emerge. Mixl1 also plays a role in closing regions of the genome that harbour potential binding sites for T/Brachyury, and the pluripotency factors Pou5f1, Sox2 and Nanog. Moreover, we have found Mixl1 timing to be instrumental in endoderm differentiation. Together, these results suggest that early Mixl1 activation makes regions of the genome inaccessible for the binding of pluripotency factors and pushes cells towards endodermal progenitors within the APS.