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 range of proteins.