Adipose tissue is an easy, accessible, and abundant source of mesenchymal stem cells (MSCs) for the reconstruction and addition of soft tissue and for restoration of soft-tissue defects associated with trauma, tumor resections, and congenital deformities. A stable source of adipose cells or tissue is needed for autologous grafting. Therefore, the aim of this study was to obtain enough autologous adipose tissue for possible clinical applications. For this purpose, we isolated MSCs (CD34+/CD90+) from human lipoaspirated or resected fat, which differentiated into adipocytes when placed in culture. Human adipose tissue is a paramount source of autologous MSCs were capable of generating a complete adipose tissue in vitro. Differentiated adipocytes expressed a strong positivity for several specific antibodies, including adiponectin and peroxisome proliferator-activated receptor gamma. In addition, fibroblasts (approximately 10% of the whole sorted-cell population) started to secrete an extracellular matrix after 60 days that was strongly positive for type I collagen and fibronectin. After long-term culture (4 months), an adipose tissue with collagenic fibers and vessels was obtained. This tissue was comparable with adult human adipose tissue and therefore may be a criterion standard for future tissue repair and regeneration and for therapeutic and transplantation purposes.