Hydrogels have been widely used to mimic the biochemical and mechanical environments of native extracellular matrices for cell culture and tissue engineering. Among them, self-assembling peptide hydrogels are of special interest thanks to their great biocompatibility, designability and convenient preparation procedures. In pioneering studies, self-assembling peptide hydrogels have been used for the culture of bone marrow cells. However, the low mechanical stability of peptide hydrogels seems to be a drawback for these applications, as bone marrow cells prefer hard substrates for osteogenic differentiation. In this work, we explored the use of hydroxyapatite (HAP)-peptide hybrid hydrogels for three-dimensional (3D) culture and differentiation of osteogenic MC3T3-E1 cells. We used HAP nanoparticles as crosslinkers to increase the mechanical stability of peptide hydrogels. Meanwhile, HAP provided unique chemical cues to promote the differentiation of osteoblasts. A phosphate group was introduced to the self-assembling peptide so that the peptide fibers could bind to HAP nanoparticles specifically and strongly. Rheological characterization indicated that the hybrid hydrogels were mechanically more stable than the hydrogels containing only peptides and can be used for long term cell culture. Moreover, the hydrogels were biocompatible and showed very low cytotoxicity. The favorable mechanical properties of the hybrid hydrogels and the chemical properties of HAP synergistically supported the differentiation of MC3T3-E1 cells. Based on these characterizations, we believe that these hybrid hydrogels can potentially be used as scaffolds for cartilage and bone regeneration in the future.