GATA1 and NF-E2 p45 are two important regulators of megakaryopoiesis. Whereas GATA1 is known to regulate the p45 gene, details of the GATA1 contribution to the spatiotemporal expression of the p45 gene remain to be elucidated. To clarify the relationship between GATA1 and p45, we performed genetic complementation rescue analysis of p45 function in megakaryocytes utilizing the hematopoietic regulatory domain of the Gata1 gene (G1HRD). We established transgenic mouse lines expressing p45 under G1HRD regulation and crossed the mice with p45-null mice. Compound mutant mice displayed normal platelet counts and no sign of hemorrhage, indicating that G1HRD has the ability to express p45 in a spatiotemporally correct manner. However, deletion of 38 amino acids from the N-terminal region of p45 abrogated the p45 rescue function, suggesting the presence of an essential transactivation activity in the region. We then crossed the G1HRD-p45 transgenic mice with megakaryocyte-specific Gata1 gene knockdown (Gata1ΔneoΔHS) mice. The G1HRD-p45 transgene was insufficient for complete rescue of the Gata1 ΔneoΔHS megakaryocytes, suggesting that GATA1 or other factors regulated by GATA1 are required to cooperate with p45 for normal megakaryopoiesis. This study thus provides a unique in vivo validation of the hierarchical relationship between GATA1 and p45 in megakaryocytes.