MicroRNAs (miRNAs), short non-coding RNAs of ~ 21 to 23 nucleotides in length, regulate target mRNAs post-transcriptionally. They play an important role in many different cellular, developmental, and physiological processes including hematopoiesis. It was the aim of this study to characterize the miRNA expression in human hematopoietic stem and progenitor cells. Hematopoietic stem cells (HSCs) have the ability to generate all different kinds of blood cells and express a number of specific surface markers such as CD34 or CD133. As CD133+ cells appear to be ancestral to CD34+ cells, the miRNA profile of these cells could broaden the knowledge of the miRNA role in the differentiation of CD133+ cells. To analyze the function of miRNAs in HSCs, a miRNA microarray platform (miRXploreTM) was developed. The platform was used, inter alia, to validate sequencing data of Small RNA libraries. Standard array experiments measure relative expression levels. However, absolute expression levels in terms of copy numbers per cell are also highly relevant. Therefore, a method for absolute quantification of miRNAs that relies on a universal reference – an equimolar pool of about 1000 synthetic miRNAs of known concentration – was developed. To characterize the role of miRNAs in hematopoiesis, different bone marrow subpopulations, namely CD133+, CD34+CD133– and CD34–CD133– cells, were isolated by magnetic cell separation. The analysis revealed 18 significantly differentially expressed miRNAs between CD133+ and CD34+CD133– cells, that could be validated via qRT-PCR and Solexa sequencing. To further analyze the role of the differentially expressed miRNAs in CD133+ stem cells, mRNA expression profiles were generated and the coexpression of bioinformatically predicted miRNA-mRNA pairs was examined. Luciferase assays were established to validate the predicted targets under physiological conditions. The miRNA-mRNA interactions that could be validated were miR 142-3p and CD133, miR-29a and FZD5 as well as miR-29a and TPM1. TPM1, an actin binding protein, and FZD5, a receptor of the Wnt-signaling pathway, play a role in the remodelling of the cytoskeleton. Further analysis of the predicted miRNA targets revealed that the miRNA targets are enriched for Gene Ontoloy categories related to stem cell-relevant processes. The differentially expressed miRNAs probably prevent differentiation of CD133+ cells and have an anti-apoptotic effect. Furthermore, first experiments were performed to analyze the influence of the differentially expressed miRNAs on the cultivation of CD133+ cells. In vitro expansion of CD133+ cells has turned out to be difficult as most of the tested culture supplements can induce proliferation but are unable to prevent differentiation. The addition of miRNAs to the culture medium could lead to an expansion of CD133+ cells without losing the primitive phenotype. Therefore, the influence of miRNA transfections on CD133+ cells was analyzed in a first step. In conclusion, the generated miRNA signature of CD133+ is the first comprehensive characterization of hematopoietic progenitor cells on miRNA level and will be highly relevant for the application of miRNAs in the field of regenerative medicine