We engineered an acellular biomimetic microenvironment to regulate stem cell fate and applied it to maintain mouse embryonic stem (ES) cell self-renewal. In the 3D environment formed using hydrogel scaffolds in which specific integrin ligation was provided. Stat3 activation by exogenous leukemia inhibitory factor (LIF) no longer acted as a limiting factor for stem cell self-renewal. Instead, simultaneous stimulation of integrins alpha(5)beta(1), alpha(v)beta(5), alpha(6)beta(1) and alpha(9)beta(1) within the 3D scaffold greatly increased Akt1 and Smad 1/5/8 activation, which resulted in prolonged self-renewal of the ES cells. The ES cells exposed to the combined stimulation of the integrins for 4 wk in LIF-free 3D scaffolds maintained the spherical morphology of cell colonies without losing any activity of pluripotency. In conclusion, cell niche-specific integrin signaling within the 3D environment supported mouse ES cell self-renewal, and the resulting integrin signaling replaced Stat3 with Akt1 and Smad 1/5/8 as critical signals for mouse ES cell self-renewal. (c) 2012 Elsevier Ltd. All rights reserved.