Dendrigrafts are multivalent macromolecules with less ordered topology and higher branching than dendrimers. Exhibiting a high density of terminal amines, poly-L-lysine dendrigrafts of the fifth generation (DGL G5) allow hydrogel formation with tailorable crosslinking density and surface modification. This work presents DGL G5 as multifunctional crosslinkers in biomimetic PEG hydrogels to favour the osteogenic differentiation of human mesenchymal stem cells (hMSCs). DGL G5 reaction with dicarboxylic-acid PEG chains yielded amide networks of variable stiffness, measured at the macro and surface nanoscale. Oscillatory rheometry and compression afforded consistent values of Young's modulus, increasing from 8 to more than 30 kPa and correlating with DGL G5 concentration. At the surface level, AFM measurements showed the same tendency but higher E values, from approximately 15 to more than 100 kPa, respectively. To promote cell adhesion and differentiation, the hydrogels were functionalised with a GRGDSPC peptide and a biomimetic of the bone morphogenetic protein 2 (BMP-2), ensuring the same grafting concentrations (between 2.15 ± 0.54 and 2.28 ± 0.23 pmols mm-2) but different hydrogel stiffness. 6 h after seeding on functionalised hydrogels in serum-less media, hMSC showed nascent adhesions on the stiffer gels and greater spreading than on glass controls with serum. After two weeks in osteogenic media, hMSC seeded on the stiffer gels showed greater spreading, more polygonal morphologies and increased levels of osteopontin, an osteoblast marker, compared to controls, which peaked on 22 kPa-gels. Together, these results demonstrate that DGL G5-PEG hydrogel bioactivity can influence the adhesion, spreading and early commitment of hMSCs.