CD133 (prominin-1) was the first in a class of novel pentaspan membrane proteins to be identified in both humans and mice, and was originally classified as a marker of primitive haematopoietic and neural stem cells. Due to the highly restricted expression of CD133 family molecules on plasma membrane protrusions of epithelial and other cell types, in association with membrane cholesterol, a role in the organization of plasma membrane topology has also recently been assigned to this family. Studies have now confirmed the utility of CD133 as a marker of haematopoietic stem cells for human allogeneic transplantation. In addition, CD133 represents a marker of tumour-initiating cells in a number of human cancers, and therefore it may be possible to develop future therapies towards targeting cancer stem cells via this marker. The development of such therapies will be aided by a clearer understanding of the molecular mechanisms and signalling pathways that regulate the behaviour of CD133-expressing cells, and new data outlining the role of Wnt, Notch, and bone morphogenetic protein (BMP) signalling in CD133(+) cancer stem cell regulation are discussed within.