Adipose tissue-derived mesenchymal stem cells (ASCs) have become an increasingly interested cell source for the scientists in the fields of stem cell biology and regenerative medicine. ASCs have already been used in a number of clinical trials, and some successful outcomes have been reported in bone tissue regeneration. Here we describe the protocols which mimic the factors in bone healing microenvironment, including inflammation burst, osteoblasts, and bone biomimetic scaffolds to direct ASCs into osteogenic differentiation.