Naïve CD4+ T helper cells can be induced to differentiate towards at least four types of committed helper T cells, namely T helper 1 (Th1), Th2, Th17 and regulatory T cells (Treg) according to the local cytokine milieu. The committed cells are characterized by the expression of specific transcription factors and by the cytokines released. Th17 cells are very efficient inducers of tissue inflammation and crucial initiators of organ specific autoimmunity. FoxP3+ Treg cells are necessary and sufficient to prevent autoimmunity. There is a reciprocal relationship between pathogenic Th17 cells and Foxp3+ Treg cells. Therefore the need to simultaneously detect FoxP3 and IL-17 is becoming increasingly important. Since mouse FoxP3 is sensitive to fixation buffer, it is important to use the appropriated buffers and protocols for its detection. In this report, we described the development of a fix/perm buffer system and protocol for simultaneous detection of mouse FoxP3 and IL-17 with cell surface markers CD4 and CD25. Using the optimized buffer and storage conditions, staining can be done on freshly fixed cells or stored fixed cells. In addition, effects of temperature during the fix/perm steps and the sensitivity of certain clones to the fixation process will be discussed.