Pericytes represent important support cells surrounding microvessels found in solid organs. Emerging evidence points to their involvement in tumor progression and metastasis. Although reported to be present in the human lung, their specific presence and functional orientation within the tumor microenvironment in non-small cell lung cancer (NSCLC) has not yet been adequately studied. Using a multiparameter approach, we prospectively identified, sorted and expanded mesenchymal cells from human primary NSCLC samples based on co-expression of CD73 and CD90 while lacking hematopoietic and endothelial lineage markers (CD45, CD31, CD14 and Gly-A) and the epithelial marker EpCAM. Compared to their normal counterpart, tumor-derived Lineage-EpCAM-CD73+CD90+ cells showed enhanced expression of the immunosuppressive ligand PD-L1, a higher constitutive secretion of IL-6 and increased basal αSMA levels. In an in vitro model of 3D microvessels, both tumor-derived and matched normal Lineage-EpCAM-CD73+CD90+ cells supported the assembly of perfusable vessels. However, tumor-derived Lineage-EpCAM-CD73+CD90+ cells led to the formation of vessels with significantly increased permeability. Together, our data show that perivascular-like cells present in NSCLC retain functional abnormalities in vitro. Perivascular-like cells as an eventual target in NSCLC warrants further investigation.