Giant cell tumor (GCT) derived stromal cells (GCTSCs) have been identified as the neoplastic cell population of GCTs. Within these stromal cells a subpopulation has been identified that shares several features with mesenchymal stem cells (MSCs) indicating that these neoplastic cells develop from MSCs. Although spontaneous transformations of MSC have already been observed in vitro and in vivo the underlying molecular mechanisms are poorly understood. As microRNAs are crucially involved in tumorigenesis and the modulation of stem cell fate and behavior, they represent promising candidates for the regulation of this process. Therefore, the aim of this study was the comparative analysis of the microRNA expression profiles of GCTSCs and MSCs in order to identify differentially expressed microRNAs and their target genes. We could identify a microRNA signature consisting of 26 differentially expressed microRNAs that perfectly separates these two cell types. One of the microRNAs with the most pronounced differences in expression levels was miR-224. We could confirm the already known regulation of the apoptosis inhibitor API5 by miR-224 and could further identify three novel miR-224 target genes (SMAD5, SLMAP, H3.3B). The involvement of these genes in the regulation of apoptosis resistance, proliferation, differentiation and the regulation of gene transcription suggests pivotal roles of these genes in the neoplastic transformation of MSCs during GCT development.