The Microphthalmia-associated transcription factor (Mitf) is an essential basic helix-loop-helix leucine zipper transcription factor for mast cell development. Mice deficient in Mitf harbor a severe mast cell deficiency, and Mitf-mutant mast cells cultured ex vivo display a number of functional defects. Therefore, an understanding of the genetic program regulated by Mitf may provide important insights into mast cell differentiation. Multiple, distinct isoforms of Mitf have been identified in a variety of cell types; we found that Mitf-a, Mitf-e, and Mitf-mc were the major isoforms expressed in mast cells. To determine the physiologic function of Mitf in mast cells, we restored expression of these isoforms in primary mast cells from Mitf ؊/؊ mice. We found that these isoforms restored granular morphology and integrin-mediated migration. By microarray analysis, proteases, signaling molecules, cell surface receptor, and transporters comprised the largest groups of genes up-regulated by all isoforms. Furthermore, we found that isoforms also regulated distinct genes sets, suggesting separable biological activities. This work defines the transcriptome regulated by Mitf in mast cells and supports its role as master regulator of mast cell differentiation. Expression of multiple isoforms of this transcription factor may provide for redundancy of biological activities while also allowing diversity of function. (2), congestive heart failure (3), and multiple sclerosis (4). Although mast cells have the capacity to promote diverse pathologic states, they also participate in normal physiologic processes, such as innate immunity against parasitic and bacterial infections (5, 6). These diverse functions may be regulated by distinct and shared pathways. A dissection of these pathways may allow selective regulation of these processes for therapeutic benefit.A number of transcription factors play essential roles in mast cell development, including the zinc finger factors GATA-1 (7) and GATA-2 (8), the Ets family member PU.1 (9), and the helixloop-helix leucine zipper factor, Microphthalmia-associated transcription factor (Mitf) 4 (10, 11 To date, nine distinct isoforms of Mitf have been identified (22). These isoforms arise from an alternative splicing event that joins a unique first exon with the common body of the gene. The resulting proteins possess distinct amino termini, but share transactivation, DNA binding, and dimerization motifs. The expression of alternatively spliced transcripts may be controlled by distinct promoter/ enhancer elements, allowing for differential regulation of isoforms. Furthermore, transcripts that encode for unique proteins species may possess distinct biological functions. Alternative splicing of mRNA transcripts is a widespread mechanism for generating diversity in multicellular organisms. Thirty-five to 65% of human genes are predicted to undergo alternative splicing, the majority of which encode for distinct protein species (23). This mechanism is particularly widespread in complex systems such ...