Multilayers of poly-l-lysine/poly-l-glutamic acid (PLL/PLGA) were constructed by layer-by-layer deposition on an end-tethered cationic PLL brush film serving as an initial layer. Increasing the number of coupling layers increased the thickness and the hydration of the films, and decreased the films' shear modulus and serum adsorption. These films were used to culture primary mouse fetal liver cells. Fetal liver stem/progenitor cells (FLSPCs) were isolated and maintained on the PLGA-terminal PLL/PLGA surfaces, forming colonies with clear boundaries that were partially attached to the surface, with cross-sectional areas of ~500 to ~2500 μm(2) after 2 days culture. Long-term studies showed that the cluster size of colonies slowly expanded and was correlated with the surface properties. For example, on the thicker films with shear modulus, G, less than 5 kPa, FLSPCs cluster size was constrained within a small distribution with less than 4000 μm(2) of projected area, whereas on the thinner films with G > 30 kPa, clusters were expanded and widely distributed, with projected areas over 4000 um(2). Immunostaining studies suggested that clusters with a small size maintained the self-renewal characteristics of stem cells, while the expanded clusters were clearly the results of spontaneous differentiation, exhibiting hepatocyte-like properties. On PLL-terminal t-(PLL/PLGA) films, which are less favorable for stem cell cultures than PLGA-terminal t-(PLL/PLGA) films, the cluster size distribution was also correlated with the film thickness, with more clusters of small size preserved on the thicker films. We observed that a soft, hydrated, serum-free surface could restrict the FLSPC expansion, resulting in self-maintenance of FLSPC colonies.