This unit describes a method of performing fluorescent in situ hybridization (FISH) of XIST and Cot-1 RNA in human pluripotent stem cells (hPSC) to characterize the epigenetic status of X-chromosome inactivation (XCI). hPSC laboratories commonly practice karyotypic analysis to monitor genetic stability; however, epigenetic stability is often overlooked. Several laboratories have recently shown that markers of XCI can be used as one effective screen to monitor the epigenetic status of hPSCs. Human embryonic stem cells (HESC) fall into three classes of XCI states: upregulating XIST upon differentiation, always expressing XIST in the undifferentiated and differentiated states, and never expressing XIST in the undifferentiated and differentiated states. Failure to express XIST represents an especially concerning state in hESC, as this state does not occur in healthy female cells but is often seen in malignancies. Herein, methods of carrying out XIST RNA and Cot-1 RNA FISH are described. FISH analysis of XIST RNA, unlike general expression analysis such as RT-PCR, allows for the classification of XCI on a single-cell level, enabling a quantitative determination of the degree of epigenetic change across the population. The complementary Cot-1 analysis measures the extent of repeat element expression throughout the nucleus and therefore enables determination, at a cytological level, of the extent to which the X chromosome is silent. Because the different steps of XCI are some of the first epigenetic changes to take place in differentiating hESC, analysis of the XCI state provides a first indication of an hESC culture's overall health.