Collagen is the main protein component of the extracellular matrix of bone, and it has structural and instructive properties. Collagen undergoes many post-translational modifications, including extensive crosslinking. Although defective crosslinking has been implicated in human syndromes (e.g., osteogenesis imperfecta or Ehlers-Danlos syndrome), it is not clear to what extent crosslinking is necessary for collagen's instructive properties during bone formation. Here we report that inhibition of collagen crosslinking in the mouse pre-osteoblast cell line MC3T3-E1 impairs the osteogenic program. Genome-wide expression profiling of beta-aminopropionitrile–treated and control cells revealed that matrix deposition by MC3T3-E1 cells provides a feed back signal, driving cells through the differentiation process, that is strongly impaired when crosslinking is inhibited. Inhibition of crosslinking did not affect osteogenic differentiation of human mesenchymal stem cells (hMSCs), shown by the expression of alkaline phosphatase and genome-wide gene expression analysis, although it enhances matrix mineralization. In conclusion, collagen crosslinking harbors instructive properties in MC3T3-E1 differentiation but plays a more-passive role in differentiation of bone marrow-derived hMSCs.