The adipose-derived stromal vascular fraction (SVF) is a promising new therapy for equine flexor tendonitis. This heterogeneous population of cells may improve tendon healing via the production of growth and chemotactic factors capable of recruiting endogenous stem cells and increasing extracellular matrix production by tendon fibroblasts (TFBL). The purpose of this study was to evaluate the ability of adipose-derived cells (ADC) culture expanded from the SVF to act as trophic mediators in vitro. We hypothesized that ADCs would produce growth and chemotactic factors important in tendon healing and capable of inducing cell migration and matrix protein gene expression. Superficial digital flexor tendons and adipose tissue were harvested from eight adult horses and processed to obtain SVF cells, ADCs and TFBLs. Adipose-derived cells and TFBLs were grown in monolayer culture for growth factor quantification, to produce conditioned media for microchemotaxis, and in co-culture for quantification of matrix protein gene expression by TFBLs. Growth factor gene expression by SVF cells was significantly greater than in ADCs or TFBLs. Co-culture of TFBLs and ADCs resulted in modest up-regulation of matrix protein expression (collagen types I and III, decorin, and cartilage oligomeric matrix protein) by TFBLs. Media conditioned by ADCs induced ADC migration in a dose dependent manner. These findings support the role of both SVF and ADCs as trophic mediators in tendon regeneration. The differences detected in gene expression between SVF cells and ADCs indicate that additional studies are needed to evaluate the changes that occur during culture of these cells.