Enzymatic digestion, the commonly used method of adipose-derived stromal cells isolation, is time consuming and expensive, especially when applied to large volumes of tissue. In the present study, the characteristics of the cells obtained by adipose tissue explant culture were studied. We found that adipose tissue fragments could adhere onto the growth surface of flasks in a very short time after plating and that fibroblast-like cells migrated from the explants and reached confluence. Morphologic analysis and surface markers expression suggested the mesenchymal origin of the cells derived from adipose tissue explants. After in vitro expansion these cells were successfully induced into adipogenic, osteogenic, and chondrogenic lineages, which demonstrated their multipotency. The high growth rate and colony-forming efficiency of explant-derived cells were similar to those of cells obtained by digestion. Furthermore, explant culture gave higher yield of cells than digestion method after primary culture. The experiment of ectopic adipogenesis in nude mice suggested the prospects for tissue engineering of these cells. In conclusion, we obtained multipotent stromal cells from adipose tissue by explant culture, and this method was simple, time saving, and gave a high yield of cells. Therefore, explant culture can be used as an effective way to isolate adipose-derived stromal cells for tissue engineering.