Surgical reconstruction of the anterior cruciate ligament (ACL) has a protracted healing phase due to poor osseous tissue integration at the graft/host interface. Intervention with an autologous cell-based therapy using human mes-enchymal stem cells (hMSCs) derived from haemarthrotic fluid aspirated at the acute phase of injury has been postulated to accelerate healing, though until now the practicalities of this approach have not been demonstrated. hMSCs were derived by plastic adherence from haemarthrosis fluid aspirated from 20 patients presenting at clinic with acute knee injury. Patient details were recorded including age and sex of patient, injury and time between injury and aspiration. The phenotype of hMSCs was characterised by flow cytometry analysis of cell surface antigens. Differentiation potential was analysed by culturing hMSCs with different pro-differentiation stimuli to drive osteogenesis, adipogenesis and chondrogenesis. Com-parative analysis of differentiation was made by quantitative PCR for lineage-specific gene expression and quantitative biochemical analyses. hMSC derivation was independent of age, sex and time between injury and aspiration however there was a statistically significant increase in frequency of derivation from haemarthosis samples that had been aspirated from bone fracture injuries compared to soft tissue injuries. hMSCs showed differential expression of cell surface antigens and there were also significant differences in their osteogenic, adipogenic and chondrogenic responses between samples. We have demonstrated the feasibility of deriving multipotent hMSCs from haemarthrosis fluid aspirated from acute knee injuries. Further optimisation of processing and differentiation methodologies must be achieved to develop a feasible clinical treat-ment which accelerates ACL reconstruction. This study has identified challenges in the harvesting, bio-processing and characterisation of hMSCs which would be broadly applicable to the development of all autologous orthopaedic cell therapies.