The lymphatic system is pivotal for maintaining basic body functions (homeostasis, immune surveillance, and nutrient transport), but also plays a significant role in the dissemination of solid tumours. Lymphangiogenesis is driven by lymphatic endothelial progenitor cells (LEPCs) and occurs mainly in pathological states, but remains relatively obscure. To date, the lack of adequate in vitro LEPC isolation and characterization protocols has hindered lymphatic research. In order to establish a reliable source of LEPCs for use in cell therapy and tissue engineering approaches, we compared primary cells extracted from the bone marrow (BM) and vascular stromal fractions (VSF) of C57BL/6 mice on expression of Podoplanin and LYVE1 LEPC markers, both at extraction (day 0) and after two-week lymphatic differentiation protocol in vitro. Higher levels of LEPCs were found in VSF than the BM at day 0 (n=3) (1.7% of podoplanin vs 1.18% of LYVE1 for VSF; and 0.17% of podoplanin vs 0.22% of LYVE1 for BM). Differentiation of VSF-derived MSCs largely outperformed BM-derived MSCs (n=3) (approximately 50% podoplanin-positive cells for VSF and 20% podoplanin-positive cells for BM); reaching an expression maximum at day 5 with a subsequent decrease until plateauing at day 10 for both types. Immunofluorescence staining images confirmed the increase in lymphatic lineage-specific (Podoplanin, LYVE1 and CD31) markers under differentiation conditions. Hence, optimizing the isolation and differentiation protocol of LEPCs will favour the development of new therapies for lymphatic pathologies as well as permit the analysis of their lymphangiogenic capacity in two- and three-dimensional environments in vitro.