Niches for tissue-resident mesenchymal stem cells (MSCs) have been identified in many adult tissues. In particular, MSCs residing in the vascular stem cell niche came into focus: the so-called vascular wall-resident MSCs (VW-MSCs) were, based upon their anatomic location, (1) distributed throughout the adult organism, and (2) supposed to be the first line cells which could be addressed in response to a pathologic trigger acting on or in close vicinity to the vascular system. Like tissue-resident MSCs in general, VW-MSC contribute to organ integrity and harbor the capacity to suppress inflammation and promote repair during normal vessel homeostasis, although resident MSCs present in the healthy situation of an individual seems not to bear sufficient for protection or repair following injury. In contrast, injury affected MSCs could contribute to disease induction and progression. A detailed understanding of the molecular repertoire as well as of the signaling pathways controlling stem cell fate of VW-MSCs is prerequisite to understand how (1) endogenous VW-MSCs contribute to normal vessel homeostasis as well as diseases that include the vascular system, (2) a potential on-site manipulation of these cells directly within their endogenous niche could be used for therapeutically benefits, and (3) isolated and therapeutically applied VW-MSCs in terms of exogenous MSCs with superior repair capabilities might be logically more efficient to address vascular diseases than MSCs derived from other tissues. This chapter describes a straightforward protocol for the improved isolation of human VW-MSCs.