ABSTRACT Down syndrome (DS) is caused by triplication of chromosome 21 (HSA21) and is the most common genetic cause of intellectual disability with a prevalence of 1 in 750 live births. However exactly how the increase in genetic material leads to the intellectual disability is unknown. Trisomy 21 has been shown to alter gene expression patterns across the entire transcriptome in DS. One of these alterations is the downregulation of network of genes regulating the oligodendrocyte (OL) lineage. This dysregulated gene expression identifies perturbed OL production as the potential underlying cellular mechanism of the observed white matter deficit in DS. Generation of oligodendrocyte precursor cells (OPCs) is dependent on the initiation of Olig2 by the morphogen Sonic hedgehog (SHH). However, in DS both of these crucial components of OPC genesis are impaired. Trisomic cells have a decreased mitogenic response to SHH. This decreased responsiveness to SHH may alter the expression of one of its downstream gene targets, the transcription factor Olig2, whose expression is crucial for OPC specification. Once activated, Olig2 induces the expression of a complex transcriptional network that controls the differentiation and eventual maturation of OPCs into OL. In addition to the initial altered SHH-mediated activation, the triplication and mis-expression of Olig2 in trisomic cells may compound the initial transcriptional changes and further dysregulate downstream gene expression. This project aims to examine the molecular consequences of these known perturbations in trisomic cells and how they lead to changes in OPC development by differentiating isogenic pairs of euploid and trisomic induced pluripotent stem cell (iPSCs) lines derived from people with DS into neural progenitor cells (NPCs) and OPCs. In Aim 1 we will test the effect dysfunctional SHH signaling has on OPC generation in DS by increasing the activation of the SHH signaling pathway both through application of a Smoothened agonist (SAG) and shRNA knockdown of the initial SHH receptor PTCH which is overexpressed in DS. In Aim 2 we will focus on how triplication of Olig2 affects its function as a key transcription factor regulating the transcriptional network by identifying differential binding of the transcription factor between trisomic and euploid cells throughout OL development. As a direct test of the effect transcription factor triplication has on OPC related transcriptional networks, we will perform RNA-seq at different OPC developmental stages and use weighted gene co-expression analysis (WGCNA) to identify the modules that contain the genes identified via ChIP-seq differential binding and examine how these networks as a whole differ between euploid and trisomic cells. Together these aims will elucidate, for the first time, the molecular underpinnings of alterations in oligodendrocyte development that may be leading to the observed white matter deficit in DS.