In higher eukaryotes, the Retinoblastoma and E2F families of proteins control the transcription of a large number of target genes. Here, we have mutated the second Drosophila Retinoblastoma family gene (Rbf2), and contrasted the in vivo molecular functions of RBF2 with dE2F2, the only E2F partner of RBF2. Previous studies failed to uncover a unique role for RBF2 in E2F regulation. Here we find that RBF2 functions in concert with dE2F2 in vivo to repress the expression of differentiation markers in ovaries and embryos where RBF2 is highly expressed. We have compared the profiles of transcripts that are mis-expressed in ovaries, embryos and S2 cells where RBF2 function has been ablated and find that RBF2 and dE2F2 control strikingly different transcriptional programs in each situation. In vivo promoter occupancy studies point to the redistribution of dE2F/RBF complexes to different promoters in different cell types as one mechanism governing the tissue-specific regulation of dE2F/RBF target genes. These results demonstrate that RBF2 has a unique function in repressing E2F-regulated differentiation markers and that dE2F2 and RBF2 are required to regulate different sets of target genes in different tissues.