REST (RE1 silencing transcription factor), also known as NRSF (neuron-restrictive silencer factor), is well-known as a transcriptional repressor of neural genes in non-neural tissues. Dysregulation of REST activity is thought to play a role in diverse diseases including cardiac hypertrophy, Down Syndrome, Huntington's disease and cancer. Previous studies examining the pluripotency transcriptional network in mouse as well as human embryonic stem cells (hESCs) have revealed that REST is regulated by the pluripotency factors OCT4, NANOG and SOX2. The goal of the present study was to evaluate the role of REST in hESCs. An inducible REST knockdown system was used to examine growth and differentiation over short and long-term culture. Interestingly, altering REST levels in multiple hESC lines did not result in loss of self-renewal, but instead led to aneuploidy. During differentiation, reduced REST levels led to altered MAPK/ERK and WNT signaling, as well as upregulation of endoderm and mesoderm markers. Critical hurdles for the translation of the clinical potential of hESCs into practice are their tumorigenic capacity, and the inefficiency in tailoring lineage differentiation. Elucidating the role of REST in regulating cell fate and genetic stability of hESCs could enable development of robust methods to stably culture and tailor lineage differentiation of these cells for use in regenerative medicine applications.