There are observed differences in the immune system between females (XX) and males (XY). Typically, females exhibit enhanced resistance to viral and bacterial infections compared to males. At the same time, autoimmune diseases are more prevalent in females with 4 out of 5 autoimmune patients being female. Studies have shown that this variation between the sexes may be correlated with a process known as X-chromosome inactivation (XCI). XCI is a process where one X chromosome is silenced in XX females to match gene expression levels with XY males. The inactivated X chromosome (Xi) is coated with a long non-coding RNA called Xist which recruits silencing proteins that inhibit transcription. However, XCI is not a perfect mechanism and though it varies between each woman, up to 23% of X-linked genes have shown to escape XCI, exhibiting bi-allelic expression. Importantly, some of these escaped genes may contribute to the differing immune systems strengths between the sexes. Most of the research on XCI escape gene dosage relies on bulk RNA-seq data. This bulk data does not address questions about the heterogeneity of escape, that is, how XCI and escape differs cell to cell within the same population. Specifically, it has been observed that immune cells exhibit this heterogeneity though it is still unclear how that changes post stimulation. To address this research gap, we use RNA single molecule fluorescence in situ hybridization (smFISH), which binds to target RNA transcripts and allows us to quantify levels of gene expression. Sequential allele-specific DNA FISH is used to target single nucleotide polymorphisms (SNPs) of each X-chromosome to attribute each RNA transcript to its chromosome of origin. In this study, we established a pipeline to use sequential smFISH and allele-specific DNA FISH to analyze XCI escape in naïve and immune-challenged mouse immune cells.