There is increasing interest in gliogenesis as the relevance of glia to both brain development and pathology becomes better understood. However, little is known about this process. The use of multidimensional protein identification technology (MudPIT) to identify changes in phosphoprotein levels in rat neural precursor cells treated with cytokines or retinoic acid showed that phosphorylation of the catalytic subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K p110alpha) and dephosphorylation of the inositol phosphatase synaptojanin-1 were common to the gliogenic stimuli. Although PI3K was found to be involved in both neuro- and astrogliogenesis, synaptojanin-1 was specifically involved in astrogliogenesis of neural precursor cells. The role of synaptojanin-1 in astrogliogenesis was further confirmed by analysis of neuron- and glia-specific markers in synaptojanin-1 knockout mouse brain. Additional experiments showed that the Sac1-like phosphatase domain of synaptojanin-1 is responsible for the observed astrogliogenic effect. Our results strongly indicate that phosphatidylinositol metabolism plays a key role in astrogliogenesis. The relevance of our findings for Down's syndrome pathology is discussed.