--- - |2- Craniofacial defects can cause morbidness. Adipose‐derived stem cells (ADSCs) have shown great promise for osteogeneration and vascularization; therefore cocultures of differentiated ADSCs are explored to increase bone and vessel formation. In this study, ADSCs were induced into osteogenic ADSCs (os‐ADSCs) and endothelial ADSCs (endo‐ADSCs) cells, which were then cocultured in variable proportions (os‐ADSCs/endo‐ADSCs = 2:1, 1:1, 1:2). The os‐ADSCs in a ratio of 1:1 expressed more ALP, RUNX2 and COL‐I, whereas VEGF, vWF and CD31 were upregulated in the endo‐ADSCs of this group. Next generation RNA sequencing (RNA‐seq) was performed to evaluate the molecular mechanisms of cocultured ADSCs. The os‐ADSCs and endo‐ADSCs interacted with each other during osteogenic and angiogenic differentiation, especially at the ratio of 1:1, and were regulated by vascular‐related genes, cell‐mediated genes, bone‐related genes and the transforming growth factor β signaling pathway (TGF‐β), mitogen‐activated protein kinase signaling pathway (MAPK) and wnt signaling pathway (Wnt). Angptl4, apoe, mmp3, bmp6, mmp13 and fgf18 were detected to be up‐regulated, and cxcl12 and wnt5a were down‐regulated. The results showed that the gene expression levels were consistent with that in RNA‐seq. The cells were then seeded into self‐assembling peptide RADA16‐I scaffolds as cocultures (1:1) and monocultures (ADSCs, os‐ADSCs, endo‐ADSCs). The results showed that the cells of all groups grew and proliferated well on the scaffolds, and the cocultured group exhibited better osteogeneration and vascularization. In conclusion, cocultured os‐ADSCs and endo‐ADSCs at the ratio of 1:1 showed strong osteogenic and angiogenic differentiation. There is a great potential for osteogenesis and vascularization by 3D culturing cells in a 1:1 ratio in self‐assembling peptide RADA16‐I scaffolds, which requires evaluation for bone regeneration in vivo. - Journal of Cellular Physiology, Volume 233, Issue 12, Page 9458-9472, December 2018.