Three-dimensional (3D) spheroids have emerged as a powerful approach for studying ossification and bone regeneration. However, it remains unclear whether a 3D self-assembly process can initiate early osteogenic responses under physiologically relevant, induction-free conditions. In this study, we established a scaffold-free, self-assembling spheroid model using mouse jawbone periosteum-derived cells (jb-PDCs) without exogenous osteogenic inducers. Compared with monolayer cultures, spheroid formation induced the sequential upregulation of osteogenic markers, including Col1a2 at 12 h, Sp7 at 24 h, Spp1 at 48 h, and Runx2 after seven days. Transcriptomic profiling revealed significant enrichment of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling. Increased phosphorylation of the p65 subunit further confirmed NF-κB pathway activation. Pharmacological inhibition of NF-κB abolished the spheroid-associated upregulation of Col1a2, Sp7, and Spp1, whereas direct NF-κB activation in conventional monolayer cultures recapitulated this osteogenic gene expression program. Furthermore, spheroid formation was accompanied by marked F-actin disassembly. Pharmacological stabilization of actin filaments reduced both p65 phosphorylation and osteogenic gene expression. Together, these findings identify an F-actin/NF-κB mechanotransduction axis through which 3D self-assembly promotes early osteogenic gene expression in jb-PDCs. Our results suggest that cellular aggregation can intrinsically couple cytoskeletal remodeling to initiate early osteogenic responses. The inducer-free jb-PDC spheroid system also provides a useful platform for investigating the mechanobiology of early osteogenic responses and may inform the development of biomimetic strategies for craniofacial bone regeneration.