Activation of BMP (BMP4), canonical Wnt (Wnt3a and CHIR-99021) and Nodal (Activin A) pathways in WT EBs enhances mesoderm (Bra) differentiation, while endoderm (GATA4) differentiation remains low, and ectoderm (Otx2) differentiation diminishes over time. Treatment with these activators in Dvl TKO EBs does not rescue mesoderm induction. The relative level of endoderm marker expression is higher in Dvl TKO EBs compared to WT EBs treated with Wnt activators, Wnt3a and CHIR, but not BMP4 or Activin A. Dvl loss also delays the decrease in ectoderm lineage expression. Inhibition of BMP (LDN-198189), canonical and non-canonical Wnt (IWP L6) and Nodal (SB431542) pathways in WT EBs results in the suppression of mesoderm differentiation, recapitulating the Dvl TKO phenotype. Pathway inhibitor treatment in WT EBs, leads to similar trends of differentiation as Dvl TKO EBs. Endoderm differentiation is greatly enhanced in Dvl TKO EBs with LDN-193189 and IWP L6 treatment.