Considerable attention has recently been given to adipose-derived stem cells (ASCs) as an important source for differentiation to Schwann cells in the treatment of peripheral nerve injury, with considerable clinical advantages over the use of mesenchymal stem cells derived from bone marrow or autologous Schwann cells. However, the relationship between adipose donor site and differentiated ASC phenotype and function is presently unknown. This work systematically studied the differentiation of ASCs harvested from three anatomical sites: (i) subcutaneous; (ii) perinephric; and (iii) epididymal adipose tissue. We show that ASC source is a major determining factor of immunophenotype, multilineage differentiation, Schwann-cell protein expression, and paracrine ability to stimulate neuronal growth. Upregulation of S100β, glial fibrillary acidic protein (GFAP), and p75NGFR was observed in differentiated ASCs from perinephric fat tissue, while only the expression of S100β or GFAP and p75NGFR was elevated in differentiated ASCs from subcutaneous or epididymal fat tissue. Although the co-culture of differentiated ASCs with NG108-15 neuronal cells demonstrated that ASCs from each source could stimulate neurite outgrowth and number, differentiated ASCs from subcutaneous and perinephric fat versus epididymal fat were most effective, which was attributed to high-brain-derived neurotropic factor/nerve growth factor and low-neurotrophin-3 levels. Thus, ASCs can be obtained from different anatomical locations, and this determines Schwann-cell phenotype upon differentiation and extent of function. This work is therefore of relevance in local therapeutic delivery of ASCs for the repair of peripheral nerve injury, but also in the broader context of ASC use in related stem-cell therapies. © 2011 Wiley-Liss, Inc.