Understanding polarization and lumen formation during development of epithelial tubes may aid in understanding tumorigenesis. The normally empty luminal space fills with cells in various types of epithelial cancers. Acini, which are empty ball-like structures surrounded by a single layer of cells, can be induced to form in vitro by culturing mammary epithelial cells or cell lines in a reconstituted extracellular matrix (ECM). Nedvetsky et al. noted that cholera toxin, which stimulates the production of adenosine 3′,5′-monophosphate (cAMP), was routinely included in the medium used to induce acinar formation of the breast cancer epithelial cell line MCF10A. Addition of cell-permeable cAMP analogs that stimulated protein kinase A (PKA), but not those that stimulated only the cAMP-regulated guanine nucleotide exchange factor Epac, promoted acinar formation. Addition of PKA-stimulating cAMP analogs increased the abundance of the proapoptotic factor BIM and, at early times in culture, increased the number of apoptotic cells detected in the lumen. The cAMP-mediated increase in BIM abundance was not accompanied by an induction of BIM mRNA; however, culturing the cells in suspension or blocking integrin activity, which also increased the abundance of BIM, was associated with an increase in mRNA abundance. Thus, cAMP and loss of ECM contact appear to regulate BIM through distinct mechanisms. Addition of a β-adrenergic receptor agonist to stimulate cAMP formation through a more physiological pathway also enhanced the formation of MCF10A acini with empty lumen. Dissection of the downstream signaling events in cultured mammary epithelial cell lines or primary human mammary epithelial cells suggested that cAMP signaling reduced the activity of extracellular signal–regulated kinase (ERK) and stimulated the activity of the kinase AKT. The addition of PKA-stimulating cAMP analogs also affected the outer cells of the acini. In this case, the rate of polarization of the cells was increased and was associated with an accumulation of integrins with the α6 subunit at the outer periphery of the outer cells. Addition of a blocking antibody that recognizes α6 integrins prevented cAMP-induced polarization of the outer cells. This study identifies two distinct effects of cAMP signaling in acinar formation—a proapoptotic role in the inner cells and a polarization-promoting role in the outer cells.