Objective The inbred transgenic nude mice with green fluorescent protein gene (GFP nude mice) line that we established have played an important role in tracing studies of the glioma microenvironment, but no study has yet reported its mechanism and effect in treating bone resorption complications after autologous cranioplasty. Our purpose in this article is to analyze the phenotype of GFP nude mouse skull cells cultured in vitro to lay the foundation for preparing tool cells for the treatment of skull resorption. Methods We selected GFP mice within three days after birth. Under sterile conditions, bilateral parietal bones with periosteum were isolated and cut into small 1 mm² pieces, and then placed in 1640 medium with fetal bovine serum. We then incubated the samples in a 5% CO2 incubator. Those cells that crawled out of the bone slices were collected for short-term subculture and relevant analyses. Results Observation of primary (P0) and secondary (P1, P2) cells indicates that the passage time in which cells grow to cover 90% of the bottom of a 60 mm dish is about 6~ 8 days, with a sampling calculation of 2. 3×10⁶~2. 5×10⁶ cells per dish. The cell morphology is mainly fibrous, but also star-shaped and dendritic. All cells emit green fluorescence under a fluorescent microscope, which is consistent with that under a white light microscope. Marker protein detection showed that BMP-6+ osteoblasts and CD206⁺、CD68⁺ macrophages coexist in the entire cell population. Conclusions Based on our skull regeneration studies, macrophages must also be involved in maintaining environmental homeostasis, in addition to the osteoblastic progenitor cell role as a starting cell. Successfully cultured P0, P1, and P2 third-generation cells meet this need, and are all expected to be further used as a tool cell in skull regeneration research.