The differentiation of stem cells is a fundamental process in cell biology and understanding its mechanism might open a new avenue for therapeutic strategies. Using an ex vivo co-culture system consisting of human primary haematopoietic stem and progenitor cells growing on multipotent mesenchymal stromal cells as a feeder cell layer, we describe here the exosome-mediated release of small membrane vesicles containing the stem and cancer stem cell marker prominin-1 (CD133) during haematopoietic cell differentiation. Surprisingly, this contrasts with the budding mechanism underlying the release of this cholesterol-binding protein from plasma membrane protrusions of neural progenitors. Nevertheless, in both progenitor cell types, protein-lipid assemblies might be the essential structural determinant in the release process of prominin-1. Collectively, these data support the concept that prominin-1-containing lipid rafts may host key determinants necessary to maintain stem cell properties and their quantitative reduction or loss may result in cellular differentiation.