Abstract Cord blood-derived CD133+ cells have a degree of non-hematopoietic potential and express transcripts of pluripotency markers including Oct-4, Sox-2, Rex-1, and leukemia inhibitory factor (LIF) receptor, as well as markers of progenitor cells, such as HoxB4, brachyury, and nestin. Having shown by transcriptome analysis that the mouse embryonic fibroblast (MEF) cells routinely used to maintain pluripotent embryonic stem cells express transcripts of the WNT/BMP families of signaling factors, we have assessed the effects on proliferation and differentiation of CD133+ cells of medium conditioned (CM) by MEF, by NIH3T3, and by NIH3T3 cells stably expressing WNT1, WNT3a, WNT4, WNT5a, and WNT11. Cultivation of CD133+ cells in MEF-CM led to a significant increase in cell number after 7 days of culture, while WNT-1, WNT3a-, and WNT11-CM increased the cell number significantly by 14 days of culture. During this period, WNT3a-CM increased the proportion of nestin-expressing cells and increased the ratio of blast-like cells to macrophages, suggesting that these signaling molecules contribute to the maintenance of an undifferentiated, blast-like phenotype. The number of cells expressing the endothelial-related marker CD31+ was significantly increased following culture in WNT5a- and WNT11-CM, whereas the number of cells positive for von Willebrand (vW) factor was maintained during 14 days of culture only in the presence of WNT4-CM. In addition, WNT5a-CM led to increased β-catenin mRNA levels and the presence of β-catenin protein in the cytoplasm and nucleus, consistent with the activation of the WNT signaling pathway. We conclude that in vitro conditioning of CD133+ cells by media containing specific WNT signaling factors influences the non-hematopoietic potential of CD133+ cells and dynamically alters the expression of the neural stem/progenitor cell marker nestin and the endothelial-related cell surface markers CD31 and vW factor.