Female mice lacking the transcription factor C/EBPbeta are infertile and display markedly reduced estrogen (E)-induced proliferation of the uterine epithelial lining during the reproductive cycle. The present study showed that E-stimulated luminal epithelial cells of a C/EBPbeta-null uterus are able to proceed through the G1 phase of the cell cycle before getting arrested in the S phase. This cell cycle arrest was accompanied by markedly reduced levels of expression of E2F3, an E2F family member, and a lack of nuclear localization of cyclin E, a critical regulator of cdk2. An increased nuclear accumulation of p27, an inhibitor of the cyclin E-cdk2 complex, was also observed for the mutant epithelium. Gene expression profiling of C/EBPbeta-null uterine epithelial cells revealed that the blockade of E-induced DNA replication triggers the activation of several well-known components of the DNA damage response pathway, such as ATM, ATR, histone H2AX, checkpoint kinase 1, and tumor suppressor p53. The activation of p53 by ATM/ATR kinase led to increased levels of expression of p21, an inhibitor of G1-S-phase progression, which helps maintain cell cycle arrest. Additionally, p53-dependent mechanisms contributed to an increased apoptosis of replication-defective cells in the C/EBPbeta-null epithelium. C/EBPbeta, therefore, is an essential mediator of E-induced growth and survival of uterine epithelial cells of cycling mice.