AbstractNetrin‐1 has been shown to regulate the function of the EGF‐like protein Cripto‐1 (Cr‐1) and affect mammary gland development. Since Cr‐1 is a target gene of Nanog and Oct4, we investigated the relationship between Netrin‐1 and Cr‐1, Nanog and Oct4 during different stages of development in the mouse mammary gland. Results from histological analysis show that exogenous Netrin‐1 was able to induce formation of alveolar‐like structures within the mammary gland terminal end buds of virgin transgenic Cripto‐1 mice and enhance mammary gland alveologenesis in early pregnant FVB/N mice. Results from immunostaining and Western blot analysis show that Netrin‐1, Nanog and Oct4 are expressed in the mouse embryonic mammary anlage epithelium while Cripto‐1 is predominantly expressed outside this structure in the surrounding mesenchyme. We find that in lactating mammary glands of postnatal FVB/N mice, Netrin‐1 expression is highest while Cripto‐1 and Nanog levels are lowest indicating that Netrin‐1 may perform a role in the mammary gland during lactation. HC‐11 mouse mammary epithelial cells stimulated with lactogenic hormones and exogenous soluble Netrin‐1 showed increased beta‐casein expression as compared to control thus supporting the potential role for Netrin‐1 during functional differentiation of mouse mammary epithelial cells. Finally, mouse ES cells treated with exogenous soluble Netrin‐1 showed reduced levels of Nanog and Cripto‐1 and higher levels of beta‐III tubulin during differentiation. These results suggest that Netrin‐1 may facilitate functional differentiation of mammary epithelial cells and possibly affect the expression of Nanog and/or Cripto‐1 in multipotent cells that may reside in the mammary gland. J. Cell. Physiol. 216: 824–834, 2008, © 2008 Wiley‐Liss, Inc.