The requirements for bone substitute materials are multifaceted. Beside biomechanical stability, these materials should provide osteoconductive and osteoinductive properties to promote integration into the host tissue. So far, autologous bone is the only material, which combines all properties, but is naturally limited. Allogenic bone grafts have to be decellularized prior to implantation. This causes the reduction of biomechanical properties and the loss of osteoinductive qualities. High hydrostatic pressure (HHP) offers a gentle alternative for processing and supply of allogenic bone substitute materials while preserving biomechanical integrity. To determine whether osteogenic properties are retained by HHP treatment, mesenchymal stem cells (MSCs) were cultured with HHP-treated and untreated allogenic trabecular bone blocks up to 28 days. Both, gene expression and protein analysis showed that HHP-treated bone positively influenced differentiation of MSCs into osteoblasts and mineralization of bone matrix. This effect was greater in samples cultivated with HHP-treated bone blocks. The present study shows that HHP treatment does not result in the reduction of osteoinductivity, thus serving as an alternative approach for processing allogeneic bone substitute materials.