Abstract Matrix stiffness and corresponding mechano-signaling play indispensable roles in cellular phenotypes and functions. How tissue stiffness influences the behavior of monocytes, a major circulating leukocyte of the innate system, and how it may promote the emergence of collective cell behavior is less understood. Here, using tunable collagen-coated hydrogels of physiological stiffness, we show that human primary monocytes undergo a dynamic local phase separation to form highly patterned multicellular multi-layered domains on soft matrix. Local activation of the β2 integrin initiates inter-cellular adhesion, while global soluble inhibitory factors maintain the steady-state domain pattern over days. Patterned domain formation generated by monocytes is unique among other key immune cells, including macrophages, B cells, T cells, and NK cells. While inhibiting their phagocytic capability, domain formation promotes monocytes’ survival. We develop a computational model based on the Cahn-Hilliard equation, which includes combined local activation and global inhibition mechanisms of intercellular adhesion suggested by our experiments, and provides experimentally validated predictions of the role of seeding density and both chemotactic and random cell migration on pattern formation.
