In situ vascular tissue engineering (TE) aims at regenerating vessels using implanted synthetic scaffolds. An envisioned strategy is to capture and differentiate progenitor cells from the bloodstream into the porous scaffold to initiate tissue formation. Among these cells are the endothelial colonies forming cells (ECFCs) that can differentiate into endothelial cells and transdifferentiate into smooth muscle cells under biochemical stimulation. The influence of mechanical stimulation is unknown, but relevant for in situ vascular TE because the cells perceive a change in mechanical environment when captured inside the scaffold, where they are shielded from blood flow induced shear stresses. Here we investigate the effects of substrate stiffness as one of the environmental mechanical cues to control ECFC fate within scaffolds. ECFCs were seeded on soft (3.58±0.90. kPa), intermediate (21.59±2.91. kPa), and stiff (93.75±18.36. kPa) fibronectin-coated polyacrylamide gels, as well as on glass controls, and compared to peripheral blood mononuclear cells (PBMC). Cell behavior was analyzed in terms of adhesion (vinculin staining), proliferation (BrdU), phenotype (CD31, aSMA staining, and flow cytometry), and collagen production (col I, III, and IV). While ECFCs adhesion and proliferation increased with substrate stiffness, no change in phenotype was observed. The cells produced no collagen type I, but abundant amounts of collagen type III and IV, albeit in a stiffness-dependent organization. PBMCs did not adhere to the gels, but they did adhere to glass, where they expressed CD31 and collagen type III. Addition mechanical cues, such as cyclic strains, should be studied to further investigate the effect of the mechanical environment on captured circulating cells for in situ TE purposes.