The cAMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors is hormone responsive and critical for nearly all mammalian cell types. The basic helix-loop-helix (bHLH) family of transcription factors is important during the development and differentiation of a wide variety of cell types. Independent studies of the role of the bHLH protein scleraxis in testicular Sertoli cells and paraxis in muscle development using yeast-2-hybrid screens provided the novel observation that bHLH proteins can directly interact with ATF/CREB family members. Analysis of the interactions demonstrated the helix-loop-helix domain of bHLH proteins directly interacts with the leucine zipper (ZIP) region of CREB2/ATF4 to form heterodimers. The direct bHLH-CREB2 binding interactions were supported using co-immunoprecipitation of recombinant proteins. Structural analysis of bHLH and ATF4 heterodimer using previous crystal structures demonstrated the heterodimer likely involves the HLH and Zip domains and has the potential capacity to bind DNA. Transfection assays demonstrated CREB2/ATF4 over-expression blocked stimulatory actions of scleraxis or paraxis. CREB1 inhibited MyoD induced myogenic conversion of C3H10T1/2 cells. CREB2/ATF4 and scleraxis are expressed throughout embryonic and postnatal testis development, with scleraxis specifically expressed in Sertoli cells. ATF4 and scleraxis null mutant mice both had similar adult testis phenotypes of reduced spermatogenic capacity. In summary, bHLH and CREB family members were found to directly heterodimerize and inhibit the actions of bHLH dimers on Sertoli cells and myogenic precursor cells. The observations suggest a mechanism for direct cross-talk between cAMP induced and bHLH controlled cellular differentiation.