Mesoderm-derived cells, including bone, muscle, and mesenchymal stem/stromal cells (MSCs), constitute various parts of vertebrate body. Cell therapy with mesoderm specification in vitro may be a promising treatment for diseases affecting organs of mesodermal origin. Repair and regeneration of damaged organs with in vitro generation of mesoderm-derived tissues and MSCs hold a great potential for regenerative therapy. Therefore, understanding the signaling pathways involving mesoderm and mesoderm-derived cellular differentiation is important. Previous findings indicated the importance of Apelin receptor (Aplnr) signaling, during embryonic development, in gastrulation, cell migration, and differentiation. Nevertheless, regulatory role of Aplnr pathway in differentiation of mesoderm and mesoderm-derived MSCs remains unclear. In the current study, we tried to elucidate the role of Aplnr signaling during mesoderm cell migration and differentiation from mouse embryonic stem cells (mESCs). By activating and suppressing Aplnr signaling pathway via peptide, small molecule, and genetic modifications including siRNA- and shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout (KO), we revealed that Aplnr signaling not only induces migration of cells during germ layer formation but also enhances mesoderm differentiation through FGF/MAPK pathway. Antibody array and LC/MS protein profiling data demonstrated that Apelin-13 treatment enhanced cell cycle, EGFR, FGF, Wnt, and Integrin signaling pathway proteins. Furthermore, Aplelin-13 treatment improved MSC characteristics, with mesenchymal phenotype and high expression of MSC markers, and silencing Aplnr signaling components resulted in significantly reduced expression of MSC markers. Also, Aplnr signaling activity enhanced proliferation and survival of the cells during MSC derivation from mesoderm. [ABSTRACT FROM AUTHOR]