Transforming growth factor ␤ (TGF-␤) is a crucial mediator of breast development, and loss of TGF-␤-induced growth arrest is a hallmark of breast cancer. TGF-␤ has been shown to inhibit cyclin-dependent kinase (CDK) activity, which leads to the accumulation of hypophosphorylated pRB. However, unlike other components of TGF-␤ cytostatic signaling, pRB is thought to be dispensable for mammary development. Using gene-targeted mice carrying subtle missense changes in pRB (Rb1 ⌬L and Rb1 NF ), we have discovered that pRB plays a critical role in mammary gland development. In particular, Rb1 mutant female mice have hyperplastic mammary epithelium and defects in nursing due to insensitivity to TGF-␤ growth inhibition. In contrast with previous studies that highlighted the inhibition of cyclin/CDK activity by TGF-␤ signaling, our experiments revealed that active transcriptional repression of E2F target genes by pRB downstream of CDKs is also a key component of TGF-␤ cytostatic signaling. Taken together, our work demonstrates a unique functional connection between pRB and TGF-␤ in growth control and mammary gland development.