The expansion of human peripheral blood endothelial progenitor cells to obtain therapeutically relevant endothelial colony-forming cells (ECFCs) has been commonly performed on xeno-derived extracellular matrix proteins. For cellular therapy applications, xeno-free culture conditions are desirable to improve product safety and reduce process variability. We have previously described a novel fluorophore-tagged RGD peptide (RGD-TAMRA) that enhanced the adhesion of mature endothelial cells in vitro. To investigate whether this peptide can replace animal-derived extracellular matrix proteins in the isolation and expansion of ECFCs, peripheral blood mononuclear cells from 22 healthy adult donors were seeded on RGD-TAMRA-modified polystyrene culture surfaces. Endothelial colony formation was significantly enhanced on RGD-TAMRA-modified surfaces compared to the unmodified control. No phenotypic differences were detected between ECFCs obtained on RGD-TAMRA compared to ECFCs obtained on rat-tail collagen-coated surfaces. Compared with collagen-coated surfaces and unmodified surfaces, RGD-TAMRA surfaces promoted ECFC adhesion, cell spreading, and clonal expansion. This study presents a platform that allows for a comprehensive in vitro evaluation of peptide-based biofunctionalization as a promising avenue for ex vivo ECFC expansion.