Within the native microenvironment, extracellular matrix (ECM) components are thought to display a complex and heterogeneous distribution, spanning several length scales. Herein, the objective is to mimic, in vitro, the hierarchical organization of proteins and growth factors as well as their crosstalk. Photolithography technique was used to adjacently pattern geometrically defined regions of RGD and BMP-2 mimetic peptides onto glass substrates. These ECM-derived ligands are known to jointly regulate mesenchymal stem cells (MSCs) osteogenic differentiation. By manipulating the spatial distribution of dually grafted peptides, the extent of human MSCs osteogenic differentiation was significantly affected, depending on the shape of peptide micropatterns. Our data highlight the existence of a strong interplay between geometric cues and biochemical signals. Such in vitro systems provide a valuable tool to investigate mechanisms by which multiple ECM cues overlap to regulate stem cell fate, thereby contributing to the design of bioinspired biomaterials for bone tissue engineering applications.