MafG and p45 possess basic region-leucine zipper (bZip) domains and form a heterodimer called NF-E2, a key regulator of megakaryopoiesis. NF-E2 binds to the Maf recognition element (MARE) and activates transcription of many platelet genes. Since the bZip domain, which mediates DNA binding and heterodimerization, is the only functional domain established for MafG, it has been assumed that MafG is required only for p45 binding to MARE and to facilitate p45-mediated transcriptional activation. Analysis of the C-terminal region of MafG, which is distinct from the bZip domain, revealed that this region contains a nuclear matrix-targeting signal. We used a transgenic complementation rescue assay to delineate the function of the MafG C terminus in vivo. Transgenic mice expressing a mutant MafG protein lacking the C terminus (MafGΔC) were crossed into a MafG-null background. The compound mutant mice displayed severe thrombocytopenia and splenomegaly, which phenocopied p45-null mice. The MafG C terminus is essential for proplatelet formation and platelet gene activation but not for p45 binding to MARE. These results demonstrate that the MafG C terminus is required for NF-E2 function and suggest that efficient targeting of NF-E2 to a specific nuclear scaffold is important to achieve high-level activity.