Background: p45 NF-E2 is a bZIP transcription factor crucial for thrombopoiesis, as indicated by the fact that loss of p45 NF-E2 function results in dramatic embryonic lethal thrombopoietic defects, and its overexpression boosts platelet release. Objectives: We set out to identify the sequences responsible for p45 NF-E2 nuclear import, evaluate its transport mechanism and ascertain its functional significance. Methods: A series of p45 NF-E2 deletion constructs fused to green fluorescent protein (GFP) was created and their cellular localization examined in mammalian cells, with the factor responsible for nuclear import identified using an in vitro transport assay. A p45 NF-E2 derivative mutated in the nuclear targeting sequence (NLS) was generated and its biological activity compared to wild-type in luciferase assays, and proplatelet and platelet production measured in murine megakaryocytes transduced with a retroviral vector. Results: Here we show that residues 271-273 are essential for nuclear import of p45 NF-E2 in COS-7 and in primary bone marrow cells. The p45 NF-E2 NLS facilitates nuclear import specifically via importin (IMP) 7. Although within the DNA-binding domain of p45 NF-E2, the NLS is not essential for DNA-binding, but is crucial for transcriptional activation and biological activity; where, in contrast to wild-type, a mutant derivative with a mutated NLS failed to promote proplatelet and platelet production in murine megakaryocytes. Conclusions: The NLS is critical for p45 NF-E2 function, with this study being the first to demonstrate the importance of NLS-dependent nuclear import of p45 NF-E2 for platelet development.