The stress-activated protein kinase (SAPK) p38 can induce apoptosis, and its inhibition facilitates mammary tumorigenesis. We found that during mammary acinar morphogenesis in MCF-10A cells grown in three-dimensional culture, detachment of luminal cells from the basement membrane stimulated mitogen-activated protein kinase (MAPK) kinases 3 and 6 (MKK3/6) and p38alpha signaling to promote anoikis. p38alpha signaling increased transcription of the death-promoting protein BimEL by phosphorylating the activating transcription factor 2 (ATF-2) and increasing c-Jun protein abundance, leading to cell death by anoikis and acinar lumen formation. Inhibition of p38alpha or ATF-2 caused luminal filling reminiscent of that observed in ductal carcinoma in situ (DCIS). The mammary glands of MKK3/6 knockout mice (MKK3(-/-)/MKK6(+/- )) showed accelerated branching morphogenesis relative to those of wild-type mice, as well as ductal lumen occlusion due to reduced anoikis. This phenotype was recapitulated by systemic pharmacological inhibition of p38alpha and beta (p38alpha/beta) in wild-type mice. Moreover, the development of DCIS-like lesions showing marked ductal occlusion was accelerated in MMTV-Neu transgenic mice treated with inhibitors of p38alpha and p38beta. We conclude that p38alpha is crucial for the development of hollow ducts during mammary gland development, a function that may be crucial to its ability to suppress breast cancer.