Eomes directs the formation of spatially and functionally diverse extraembryonic hematovascular tissues.

During mouse gastrulation, extraembryonic mesoderm (ExEM) contributes to the extraembryonic yolk sac (YS) and allantois, both of which are essential for successful gestation. Although the genetic networks coordinating intra-embryonic mesodermal subtype specification are well studied, ExEM diversification remains poorly understood. Here, we identify that embryoid body (EB) in vitro differentiation generates distinct lineages of mesodermal cells, matching YS and allantois development. Combining in vitro and in vivo mouse models, we discover that Eomesodermin (Eomes) controls the formation of YS-fated ExEM but is dispensable for allantois formation. Furthermore, simultaneous disruption of Eomes and T impedes the specification of any YS or allantois mesoderm, indicating compensatory roles for T during allantois formation upon Eomes depletion. Our study highlights previously unrecognized functional and mechanistic diversity in ExEM diversification and endothelial development and introduces a tractable EB model to dissect the signaling pathways and transcriptional networks driving the formation of key extraembryonic tissues.