Aortic valve stenosis (AVS) occurs frequently in the elderly, which is exacerbated by valve ectopic calcification. Recently, we found that mesenchymal undifferentiated cells were rich in human aortic valve interstitial cells (HAVICs), and hematopoietic stem cell marker CD34-negative cells were higher sensitive on various calcification stimulations such as tumor necrosis factor (TNF)-α. These cells were also vascular endothelial growth factor receptor 2-positive. We aimed to investigate whether TNF-α affects the gene expression of CD34 and calcification-related proteins in HAVICs, isolated from the calcified aortic valves of AVS patients. After 8h culture of HAVICs with TNF-α (30 ng/mL), the gene expressions of bone morphogenetic protein (BMP) 2 and distal-less homeobox 5 were accelerated. In addition to CD34, gene expressions of extracellular matrix proteins tenascin X and matrix Gla protein were significantly decreased after 6 days culture of HAVICs with TNF-α, resulting in accelerated calcification. Transforming growth factor-β1, known another calcification inducing factor, did not change these gene expressions. These results suggest that TNF-α-BMP2 signaling mainly contributes to valve ectopic calcification by inhibiting CD34 expression.