From a list of protein kinases (PKs) that are newly induced upon differentiation of mouse embryonic stem cells to mesendoderm, we identified a previously uncharacterized kinase, Vlk (vertebrate lonesome kinase), that is well conserved in vertebrates but has no homologs outside of the vertebrate lineage. Its kinase domain cannot be classified into any of the previously defined kinase groups or families. Although Vlk is first expressed in E-cadherin-positive anterior visceral endoderm and mesendoderm, its expression is later confined to E-cadherin-negative mesenchyme. Vlk is enriched in the Golgi apparatus and blocks VSVG transport from the Golgi to the plasma membrane. Targeted disruption of Vlk leads to a defect in lung development and to delayed ossification of endochondral bone. Vlk(-/-) mice display neonatal lethality due to respiratory failure, with a suckling defect arising from a cleft palate. Our results demonstrate that Vlk is a novel vertebrate-specific PK that is involved in the regulation of the rate of protein export from the Golgi, thereby playing an important role in the formation of functional stroma by mesenchymal cells.