Hematopoietic stem cells (HSCs) have a unique ability to self-renew and produce differentiated progeny throughout the entire life of the human body. However, present clinical use of HSCs in regenerative medicine is largely hindered by our limited understanding of the mechanisms that direct stem cell fate both in vivo and in vitro. Genetic profiling of single stem cells is an appealing method to gain a deeper understanding of these processes by dissecting the relationship between the cellular phenotype and its gene expression signature. To do so, classical methods rely on the use of bench-top assays which either lack the required efficiency for the analysis of low-copy transcripts such as transcription factors, or the ability to capture the dynamics of a stem cell population, for instance during cell division. Here, we report a novel microfluidic device for the on-chip reverse transcription (RT) of single stem cells coupled with a chip-to-world interface that enables reliable extraction of samples for virtually any bench-top assay. The designed microfluidic module allows the trapping of single cells, their lysis and mRNA capture, followed by solid-phase cDNA synthesis using the Dynabead® system. We have successfully validated and characterized all major stages of the on-chip RT protocol and obtained single-cell nested RT-PCR readouts with an efficiency superior to bench-top assays. Live single stem cells have also been extracted and analyzed via this method, thus demonstrating that the developed device can act as much as a Lab-on-a-Chip system as a micro-manipulator for single cell microfluidic handling. Furthermore, we have laid the foundations for the integration of our RT module with on-chip cell culture for automated stem cell progeny capture during division. Such a device will help to study heterogeneities not only within cell populations, but also within the progeny of a single cell.