Expression of the c-Myc oncoprotein is affected by conserved threonine 58 (T58) and serine 62 (S62) phosphorylation sites that help to regulate c-Myc protein stability, and altered ratios of T58 and S62 phosphorylation have been observed in human cancer. Here, we report the development of 3 unique c-myc knock-in mice that conditionally express either c-Myc^WT or the c-Myc^T58A or c-Myc^S62A phosphorylation mutant from the constitutively active ROSA26 locus in response to Cre recombinase to study the role of these phosphorylation sites in vivo. Using a mammary-specific Cre model, we found that expression of c-Myc^WT resulted in increased mammary gland density, but normal morphology and no tumors at the level expressed from the ROSA promoter. In contrast, c-Myc^T58A expression yielded enhanced mammary gland density, hyperplastic foci, cellular dysplasia, and mammary carcinoma, associated with increased genomic instability and suppressed apoptosis relative to c-Myc^WT. Alternatively, c-Myc^S62A expression reduced mammary gland density relative to control glands, and this was associated with increased genomic instability and normal apoptotic function. Our results indicate that specific activities of c-Myc are differentially affected by T58 and S62 phosphorylation. This model provides a robust platform to interrogate the role that these phosphorylation sites play in c-Myc function during development and tumorigenesis. Cancer Res; 71(3); 925–36. ©2011 AACR.