Adipose tissue is an easily accessible and abundant source of stem cells. Adipose stem cells (ASCs) are currently being researched as treatment options for repair and regeneration of damaged tissues. The standard culture conditions used for expansion of ASCs contain fetal bovine serum (FBS) which is undefined, could transmit known and unknown adventitious agents, and may cause adverse immune reactions. We have described a novel culture condition which excludes the use of FBS and characterised the resulting culture. Human ASCs were cultured in the novel culture medium, which included complement protein C3. These cultures, called C-ASCs, were compared with ASCs cultured in medium supplemented with FBS. Analysis of ASCs for surface marker profile, proliferation characteristics and differentiation potential indicated that the C-ASCs were similar to ASCs cultured in medium containing FBS. Using a specific inhibitor, we show that C3 is required for the survival of C-ASCs. This novel composition lends itself to being developed into a defined condition for the routine culture of ASCs for basic and clinical applications.