Patients with germline STAT3 gain-of-function (GOF) and loss-of-function (LOF) mutations were identified by whole-exome sequencing. GOF STAT3 mutations increased the expression of downstream target genes like Suppressor of Cytokine Signaling 3 (SOCS3). Although transcriptional activity was increased, phosphorylation of STAT3 both at baseline and upon stimulation was normal. To elucidate the mechanism by which normal phosphorylation of GOF STAT3 leads to altered target gene expression and immune dysfunctions, we studies STAT3 dynamic at single cell levels with the Amnis ImageStream flow cytometer. We analyzed the ratio of similarity scores between IL-6 stimulated vs. nonstimulated nuclear and cytoplasmic localization of STAT3 at different time points. In comparison with cells carrying wild type STAT3, we found that GOF STAT3 and LOF STAT3 cells have different behaviors during prolonged IL-6 stimulation. The ratio of the wild type STAT3 cells changed very little when the cells were stimulated with IL-6 from 15 to 120 min. Interestingly, the ratio of similarity score for GOF STAT3 cells were higher at 15 min and 30 min after IL-6 stimulation, and decreased for an extended stimulation of up to 120 min. Unexpectedly, the ratio of LOF STAT3 cells were lower than the wild type and the GOF cells for the shorter stimulation times of up to 30 minutes, but it increased with the time of IL-6 stimulation. These preliminary results provide more information about STAT3 behavior in a complex biological system, which may help us understand the mechanism linked to mutated STAT3 dysfunctions.