Mammalian specification of mesoderm and definitive endoderm (DE) is instructed by the two related Tbx transcription factors (TFs) Eomesodermin (Eomes) and Brachyury sharing partially redundant functions. Gross differences of mutant embryonic phenotypes suggest specific functions of each TF. To date, the molecular details of separated lineage-specific gene-regulation by Eomes and Brachyury remain poorly understood. Here, we combine mouse embryonic and stem cell-based analyses to delineate the non-overlapping, lineage-specific transcriptional activities. On a genome-wide scale binding of both TFs overlaps at promoters of target genes, but shows specificity for distal enhancer regions, that is conferred by differences in Tbx DNA-binding motifs. The unique binding to enhancer sites instructs specification of anterior mesoderm (AM) and DE by Eomes and caudal mesoderm by Brachyury. Remarkably, EOMES antagonizes BRACHYURY gene-regulatory functions in co-expressing cells during early gastrulation to ensure the proper sequence of early AM and DE lineage specification followed by posterior mesoderm derivatives