Stem cells hold tremendous promise for gene therapy, tissue engineering, and the treatment of a diverse range of injuries and disease. However, exploiting the full potential of these pluripotent progenitor cells requires the establishment of a new set of tools capable of controlling the molecular decisions that determine whether, and how these cells differentiate. Critical transitions in stem cells are controlled via signaling pathways and subsequent transcriptional regulation. In this regard, we have previously shown that designed zinc-finger protein transcription factors (ZFP TFs) are capable of regulating the expression of targeted endogenous genes with singular specificity. In the present study, we evaluated the ability of such designed ZFP TFs to control the differentiation of embryonic stem (ES) cells. To this end, we constructed ZFP TFs that target the mouse OCT4 gene, a major regulator of ES cell pluripotency and self-renewal. We show here that introduction of an activator version of this ZFP TF resulted in increased OCT4 mRNA, and the expected concomitant effects on the expression of OCT4 responsive downstream target genes. Conversely, a repressor version of the OCT4-specific ZFP TF caused a robust reduction in OCT4 expression and the reciprocal downstream effects. Significantly, ZFP TF-dependent morphological differentiation of ES cells was also observed in a manner specific to the effector domain employed. Thus ZFP TFs are shown to be capable of controlling ES cell differentiation, raising the potential for their broad application to stem cell-based therapies.