Cerebellar granule cells (CGC) are the most abundant neurons in the mammalian brain, and an important tool for unraveling molecular mechanisms underlying neurogenesis. Math1 is a bHLH transcription activator that is essential for the genesis of CGC. To delineate the effects of Math1 on CGC differentiation, we generated and studied primary cultures of CGC progenitors from Math1/lacZ knockout mice. Rhombic lip precursors appeared properly positioned, expressed CGC-specific markers, and maintained Math1 promoter activity in vivo and in vitro, suggesting that Math1 is not essential for the initial stages of specification or survival of CGC. Moreover, the continuous activity of Math1 promoter in the absence of MATH1, indicated that MATH1 was not necessary for the activation of its own expression. After 6, but not 3, days in culture, Math1 promoter activity was downregulated in control cultures, but not in cells from Math1 null mice, thus implying that Math1 participates in a negative regulatory feedback loop that is dependent on increased levels of MATH1 generated through the positive autoregulatory feedback loop. In addition, Math1 null CGC did not differentiate properly in culture, and were unable to extend processes. All Notch signaling pathway receptors and ligands tested were expressed in the rhombic lip at embryonic date 14, with highest levels of Notch2 and Jag1. However, Math1-null rhombic lip cells presented conspicuous downregulation of Notch4 and Dll1. Moreover, of the two transcriptional repressors known to antagonize Math1, Hes5 (but not Hes1) was downregulated in Math1-null rhombic lip tissue and primary cultures, and was shown to bind MATH1, thus revealing a negative regulatory feedback loop. Taken together, our data demonstrate that CGC differentiation, but not specification, depends on Math1, which acts by regulating the level of multiple components of the Notch signaling pathway.