Transcription factor GATA2 is expressed in numerous mammalian tissues, including neural, hematopoietic, cardiovascular and urogenital systems, and yet it plays important roles in the regulation of tissue-restricted gene expression. The Gata2 gene itself is also under stringent tissue-specific control and multiple cis-regulatory domains have been identified in the Gata2 locus. In this study we sought out and then examined in detail the domains that regulate Gata2 in the midbrain. We identified two discrete domains in the Gata2 promoter that direct midbrain expression; these distal 5H and proximal 2H regulatory domains are located 3.0 and 1.9 kbp, respectively, upstream of the transcriptional initiation site. Importantly, both domains contain GATA factor binding sites. Our analyses further revealed that GATA2 is essential for Gata2 gene expression in the midbrain, whereas GATA3 is not. Both the 2H and 5H domains have the independent ability to activate Gata2 gene expression in the midbrain superior colliculus, whereas the distal-5H domain is additionally capable of activating Gata2 transcription in the inferior colliculus. These results demonstrate that two distinct regulatory domains contribute to the Gata2 gene expression in the mouse midbrain and that Gata2 midbrain transcription is under positive autoregulation.