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Three-dimensional (3D) protein-patterned scaffolds provide a more biomimetic environment for cell culture than traditional two-dimensional surfaces, but simultaneous 3D protein patterning has proved difficult. We developed a method to spatially control the immobilization of different growth factors in distinct volumes in 3D hydrogels, and to specifically guide differentiation of stem/progenitor cells therein. Stem-cell differentiation factors sonic hedgehog (SHH) and ciliary neurotrophic factor (CNTF) were simultaneously immobilized using orthogonal physical binding pairs, barnase–barstar and streptavidin–biotin, respectively. Barnase and streptavidin were sequentially immobilized using two-photon chemistry for subsequent concurrent complexation with fusion proteins barstar–SHH and biotin–CNTF, resulting in bioactive 3D patterned hydrogels. The technique should be broadly applicable to the patterning of a wide …