The therapeutic promise of stem cell biology lies in its potential for cell replacement therapies in diseases where a particular cell type or cellular function has been lost. For example, the devastating neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are characterized by a progressive loss of motor neurons and consequent paralysis. Cell replacement for diseases of the nervous system poses special challenges because many neurons lose their ability to proliferate and thus regenerate after embryonic differentiation. Strategies for replacing specific neuronal cell types with cells derived from human embryonic stem (hES) cells will require understanding the genetic programs that control hES cell differentiation. These rapidly dividing pluripotent cells undergo a major transition in gene expression to become neuronal progenitor cells (NPC), while maintaining their proliferative ability. A second dramatic change in the program of gene expression occurs when these progenitor cells cease cell division and differentiate into neurons. A great deal of work has described the DNA level changes affecting gene expression in ES cells and during their transition to NPC and neurons. However, the gene expression pathway is also highly regulated at the RNA level, as the DNA gene is transcribed into RNA and the RNA then translated into protein. An important regulatory step in this pathway is the processing of the primary RNA transcript from the gene into an mRNA that can be translated into protein. This processing includes pre-mRNA splicing, and alternative splicing patterns in the pre-mRNA determine the final protein output of most human genes. Little is known about how this step in the gene expression pathway is regulated in ES cells or during their differentiation. Yet ALS and SMA can both be caused by the loss of components of the splicing machinery and a great deal of work is examining how splicing might be disrupted in mature neurons of ALS and SMA patients. In this study, we have examined how two important splicing regulators, the polypyrimidine tract binding protein (PTBP1) and its neuronal homolog PTBP2, affect splicing in normal ES and NP cells, and have characterized their regulatory networks. We adapted and applied two new technologies for RNA analysis. The first used high density RNA sequencing (RNAseq) to examine the whole transcriptome of each cell under study. From this data, we extracted information on all the splicing changes occurring during a developmental transition. The second method called CLIP identifies the sites of binding by PTBP1 and PTBP2 in the RNA of each cell type. These methods were applied to hESC, NPC and motor neurons that we have derived in culture. From this work, we identified many new genetic events that determine how ES cells differentiate into neurons. These alternative splicing events controlled by the PTB proteins affect numerous functions essential to the development and survival of neurons. We are now examining several of new genetic regulatory events in more detail to understand how pre-mRNA splicing controls cell function in normal development and in disease.