Foxp3+ CD25+ regulatory T cells (Tregs) are a rare subset within the CD4+ T cell compartment, playing a vital role in immune system regulation. Because of their immunosuppressive ability, Tregs are an attractive potential new tool for treating autoimmune diseases and preventing graft rejection. Although the population of Treg cells is well-defined by the intracellular expression of the transcription factor Forkhead box protein 3 (Foxp3), no defined surface marker distinguishes Tregs from other T cells, making reliable phenotypic characterization and isolation challenging. Furthermore, most isolation and expansion protocols are designed for human Tregs, representing a significant drawback since mice are important model organisms in the preclinical phase of drug development. A range of methodologies was employed. Treg cells were isolated from mouse spleens using immunomagnetic separation. Consequently, cells were activated and expanded for 15 days, using different ratios of Treg cells-to-activation beads in the presence of mouse interleukin-2 (mIL-2) cytokine. After the staining for viability, surface, and intracellular markers, the phenotype of Tregs and other T cell subsets was analyzed using flow cytometry (FC). To investigate the transcriptional profiles of subpopulations within the Tregs samples, we performed the 10x Genomics single-cell RNA sequencing (scRNA-seq). This study resulted in the successful establishment of isolation and expansion protocols for mouse Foxp3+ CD25+ Tregs, as well as comprehensive FC staining panels. The phenotype and transcriptome analysis revealed the heterogeneity of the Treg population already upon isolation from secondary lymphatic tissue. ScRNA-seq analysis provided a deeper insight into the Treg cells' transcriptome and revealed other important biomarkers for contaminants-free isolation and improved phenotypic characterization of Treg cells. The obtained results pave the way for developing innovative therapeutic approaches for autoimmune diseases, graft rejection and cancer, which hold a promise to greatly improve patients´quality of life.