The extracellular matrix (ECM) shapes the stem cell fate during differentiation by exerting relevant biophysical cues. However, the mechanism of stem cell fate decisions in response to ECM-backed complex biophysical cues has not been fully understood due to the lack of versatile ECMs. Here, we designed two versatile ECMs using colloidal self-assembly technology to probe the mechanisms of their effects on mechanotransduction and stem cell fate regulation. Binary colloidal crystals (BCC) with a hexagonally close-packed structure, composed of silica (5 μm) and polystyrene (0.4 μm) particles as well as a polydimethylsiloxane-embedded BCC (BCCP), were fabricated. They have defined surface chemistry, roughness, stiffness, ion release, and protein adsorption properties, which can modulate the cell adhesion, proliferation, and differentiation of human adipose-derived stem cells (hASCs). On the BCC, hASCs preferred osteogenesis at an early stage but showed a higher tendency toward adipogenesis at later stages. In contrast, the results of BCCP diverged from those of BCC, suggesting a unique regulation of ECM-dependent mechanotransduction. The BCC-mediated cell adhesion reduced the size of the focal adhesion complex, accompanying an ordered spatial organization and cytoskeletal rearrangement. This morphological restriction led to the modulation of mechanosensitive transcription factors, such as c-FOS, the enrichment of transcripts in specific signaling pathways such as PI3K/AKT, and the activation of the Hippo signaling pathway. Epigenetic analyses showed changes in histone modifications across different substrates, suggesting that chromatin remodeling participated in BCC-mediated mechanotransduction. This study demonstrates that BCCs are versatile artificial ECMs that can regulate human stem cells' fate through unique biological signaling, which is beneficial in biomaterial design and stem cell engineering.