OBJECTIVETo investigate whether CD34+ purified human muscle‐derived cells (hMDCs) are capable of multiple lineage differentiation.MATERIALS AND METHODSThe hMDCs were isolated from human skeletal muscle and purified using a CD34+ cell selection system (Dynal Biotech, Oslo, Norway). Adherent populations of cells were expanded in culture and cell differentiation was induced using different kinds of growth factors and different differentiation‐conditional media. The immunohistochemical properties of CD34+ hMDCs were examined after varying periods in culture. Reverse transcription‐polymerase chain reaction (RT‐PCR) and Western blotting were used to investigate the gene expression of the undifferentiated and differentiated hMDCs.RESULTSUsing special differentiation conditions the CD34+ hMDCs could be differentiated into myogenic cells, adipocytes, osteocytes and chondrocytes. The differentiation was confirmed by immunohistochemistry. RT‐PCR and Western blotting showed multiple‐lineage gene‐level expression in the different cultivation periods of the differentiated cells.CONCLUSIONSWe confirmed the multi‐lineage capacity of a population of stem cells, termed CD34+ hMDCs. Our findings showed that CD34+ hMDCs are capable of multiple mesodermal‐lineage differentiation, as shown by the expression of several lineage‐specific genes. They can be differentiated toward the myogenic, osteogenic, adipogenic and chondrogenic lineages. These cells might have potential for use in tissue regeneration.