The adipose tissue contains a large numbers of stem cells; adipose-derived stem cells (ADSCs) can be employed in regenerative medicine. This study was aimed at isolating ADSCs and evaluating the safety of ADSCs in mouse models. Stromal vascular fraction (SVF) was collected from the adipose tissue using collagenase. ADSCs were then isolated from SVFs by in vitro culture. The stemness of the ADSCs was evaluated in vitro based on their self-renewal potential, potential to differentiate into osteoblasts, and adipocytes, and the expression of specific markers. Finally, the tumor formation ability of ADSCs was evaluated in vivo in athymic mice. Results showed that 100% of the ADSC samples developed well and maintained homogeneity up to passage 10. The ADSCs were completely sterilized and could not form tumors in athymic mice. These initial results showed that ADSCs were safe for use in stem cell therapy.