Background: Cells possess a specialized machinery through which they can sense physical as well as chemical alterations in their surrounding microenvironment that affect their cellular behavior. Aim: In this study, we aim to establish a polyelectrolyte multilayer system of 24 layers of poly-l-lysine and hyaluronic acid to control stem cell response after chemical cross-linking. Methods and results: The multilayer build-up process is monitored using different methods, which show that the studied polyelectrolyte multilayer system grows exponentially following the islands and islets theory. Successful chemical cross-linking is monitored by an increased zeta potential toward negative magnitude and an extraordinary growth in thickness. Human adipose–derived stem cells are used here and a relationship between cross-linking degree and cell spreading is shown as cells seeded on higher cross-linked polyelectrolyte multilayer show enhanced spreading. Furthermore, cells that fail to establish focal adhesions on native and low cross-linked polyelectrolyte multilayer films do not proliferate to a high extent in comparison to cells seeded on highly cross-linked polyelectrolyte multilayer, which also show an increased metabolic activity. Moreover, this study shows the relation between cross-linking degree and human adipose–derived stem cell lineage commitment. Histological staining reveals that highly cross-linked polyelectrolyte multilayers support osteogenic differentiation, whereas less cross-linked and native polyelectrolyte multilayers support adipogenic differentiation in the absence of any specific inducers. Conclusion: Owing to the precise control of polyelectrolyte multilayer properties such as potential, wettability, and viscoelasticity, the system presented here offers great potential for guided stem cell differentiation in regenerative medicine, especially in combination with materials exhibiting a defined surface topography.