Contact guidance has been extensively explored using patterned adhesion functionalities that predominantly mimic cell–matrix interactions. Whether contact guidance can also be driven by other types of interactions, such as cell−cell adhesion, still remains a question. Herein, this query is addressed by engineering a set of microstrip patterns of (i) cell–cell adhesion ligands and (ii) segregated cell–cell and cell–matrix ligands as a simple yet versatile set of platforms for the guidance of spreading, adhesion, and differentiation of mesenchymal stem cells. It was unprecedently found that micropatterns of cell–cell adhesion ligands can induce contact guidance. Surprisingly, it was found that patterns of alternating cell–matrix and cell–cell strips also induce contact guidance despite providing a spatial continuum for cell adhesion. This guidance is believed to be due to the difference between the potencies of the two adhesions. Furthermore, patterns that combine the two segregated adhesion functionalities were shown to induce more human mesenchymal stem cell osteogenic differentiation than monofunctional patterns. This work provides new insight into the functional crosstalk between cell–cell and cell–matrix adhesions and, overall, further highlights the ubiquitous impact of the biochemical anisotropy of the extracellular environment on cell function.