The mammary gland development is regulated by steroid and peptide hormones and occurs mainly after birth, compared to the majority of the other organs in the body. The principal hormones responsible for the breast development are the estrogen, the progesterone, the growth hormone, the epidermal growth factor and the prolactin. The latter one is of specific interest since it is required for further development of the gland through pregnancy and lactation. During pregnancy and lactation, alveolar structures develop and differentiate into secretory cells. The signal transducer and activator of transcription 5 (Stat5) is known to be the main effector of prolactin signaling during pregnancy, inducing alveologenesis and differentiation of epithelial cells into secretory cells. I investigated if Stat5 has a role before pregnancy. Protein expression as well as mRNA expression analysis of glands from ovariectomized mice which were artificially stimulated with ovarian hormones (estrogen and progesterone) and control vehicle showed evidences that Stat5 expression can be stimulated by these two hormones. Contrary to the role established until now for stat5 in the mammary gland, I found by fluorescence visualization of Stat5-null epithelium as well as whole mount staining of the same glands that Stat5 is required even before pregnancy since null epithelium showed highly reduced growth already in 3 weeks-old virgin mice. This may indicate a new role for this protein in cell proliferation or survival in early stages of mouse mammary development. Furthermore, transplantation experiment of Stat5-null epithelium into wild-type recipient mice showed that pregnancy state was not able to rescue the normal phenotype. This may be due to complete loss of these cells which happened during the 5 weeks left for the transplanted epithelium to expand the cleared fat pad. This discovery opens a new debate on Stat5 role in the mammary gland development.