Mesoderm-specific transcript (Mest) is a distinct gene associated with adipocyte differentiation and proliferation. The mechanisms regulating expression of the Mest gene are not established. Therefore, we investigated Mest gene expression during adipogenic differentiation in murine 3T3-L1 preadipocytes and adipose-derived stromal cells (ADCs) from C57BL/6J mouse adipose tissue. Expression of Mest mRNA increased significantly in 3T3-L1 cells during differentiation. Additionally, Mest mRNA expression levels were additively enhanced by the inhibition of DNA methylation. Expression levels of the Mest gene were also markedly elevated in differentiating ADCs in vitro. Additionally, we showed that Mest mRNA can be upregulated by increasing intracellular cAMP, and that Mest expression is suppressed by inhibition of protein kinase A (PKA). Mest expression was regulated through cAMP-dependent PKA pathways during differentiation of preadipocytes into adipocytes in vitro, supporting the critical role of Mest in proliferation and differentiation of adipocytes.