Microfluidic channels functionalized with adhesive ligands are versatile platforms for cell separation in a variety of applications. However, not much is known about how the adhesiveness of targeted cell types can vary within such channels due to the combined influence of fluid shear forces and exposure to ligands. Using microfluidic channels and the tetrapeptide ligand arg-glu-asp-val (REDV), we demonstrate how such dynamic changes can provide a basis for the identification of three distinct phenotypes of endothelial cells: human umbilical vein endothelial cells (HUVECs), human microvascular endothelial cells (HMVECs), and endothelial colony forming cells (ECFCs). This distinction is accomplished by characterizing changes in the adhesion profiles of the three cell types in REDV-coated microfluidic channels induced by soluble REDV and fluid shear forces. The significance of this technique lies in the ability to distinguish very similar cell-types without fluorescent label-based staining or flow cytometry.