Cardiovascular calcification is one of the leading reasons of morbidity and mortality in Western countries and has many similarities to osteogenesis. The role of smooth muscle calcific transformation is well established for atherogenic lesions, but mechanisms driving initial stages of proosteogenic cell fate commitment in big vessels remain poorly understood. The role of endothelial and underlying interstitial cell interaction in driving cellular decisions is emerging from recent studies. The aim of this study was to analyze co-culture of endothelial and smooth muscle cells in vitro in acquiring proosteogenic phenotype. We co-cultured human aortic endothelial cells (EC) and human aortic smooth muscle cells (SMC) and analyzed osteogenic phenotype by ALP staining and proosteogenic gene expression by qPCR in co-cultures and in separate cellular types after magnetic CD31-sorting. In EC and SMC co-cultures osteogenic phenotype was induced as well as activated expression of RUNX2, POSTIN, BMP2/4, SOX5, COL1A SMC; co-culture of EC with SMC induced NOTCH1, NOTCH3, NOTCH4 and HEY1 expression; Notch activation by lentiviral activated Notch intracellular domain induced expression of RUNX2, OPN, POSTIN in SMC; NOTCH1 and NOTCH3 and HEY1 were selectively induced in EC during co-culture. We conclude that endothelial cells are capable of driving smooth muscle calcification via cell-cell contact and activation of Notch signaling.