The fatty layer of aspirate obtained by reaming the femoral shaft using a reamer/irrigator/aspirator (RIA) device was characterized for fatty acid content and the presence of adult stem cells. Gas chromatography analysis was performed on samples taken from multiple patients to determine and compare the fatty acid contents of aspirate lipid samples. All four patients had the same four fatty acids present in the highest percentages: oleic, palmitic, linoleic and stearic. After successful isolation from bulk material, cells isolated from this lipid-rich layer were studied to determine their osteogenic and growth potential on a clinically available ceramic bone graft substitute. The results of metabolic activity and intracellular protein assays indicated that the ceramics supported growth of the cells isolated from the aspirate fat layer, although levels of alkaline phosphatase (ALP) expression were low for cells grown on the ceramics. Cells will not transition along the osteogenic pathway when they are actively dividing, and active growth may have contributed to the lack of ALP expression in this study. Isolated cells grown on tissue culture plastic expressed significant levels of the bone marker ALP. The results of this study suggest that cells isolated from the fat layer of RIA aspirate proliferate on ceramic bone void filler and have the potential to differentiate along an osteogenic pathway. Previously considered waste, the lipid-rich fat layer of aspirate may be a source of mesenchymal stem cells that, either alone or in conjunction with currently available synthetic bone graft material, could be used to stimulate new bone growth.