Althogh Spermatogonial stem cells (SSCs) are widely studied in mice, study of pig SSCs is not sufficient for the isolation, long-term culture, and characterization. To identify the effect of growth factor in cultured pig SSC, newly generated pSSCs like cell from neonatal 5days porcine testis were cultured and investigated for the pSSCs like cell formation. Glial derived neurotrophic gactor (GDNF), fibroblast growth factor (FGF), leukemia inhibitory factor (LIF), and epidermal growth factor (EGF) were applied to culture media to identify the pSSC like cell growth and stem cell formation. The criteria for the determining of stem cell characters, morphology, number of colonies, putative stem cell marker were analysed by microspic, polymerase chain reaction (PCR) and immunocytochemistry (ICC) methods. Most of the pSSCs like cells were formed approximately 100 μm size with sphere shape. Most of the feeder cells were highly dependent on FGF that feeder cells were not stably attached on plate without FGF and colony formation of pSSC was not observed consequently. Immunocyto chemistry data revealed that this cells expressed the ubiquitin-C-terminal hydrolase 1 (UCHL-1, PGP9.5) and Dolichos Biflorus Agglutinin (DBA) in addition of 20 ng/ml EGF, 10 ng/ml FGF, 10 ng/ml GDNF, 10 U3/ml LIF. In addition, Alkaline Phosphatase ()was positive in all period of culture. This study suggest that various growth factorsinp SSC culture system is important to regulate and maintain the pSSC. In conculsion, although the precise role of growth factor in pSSC proliferation need to be clarified, combination of growth factor might be critical in order to derivation and proliferation of neonatal pSSCs and spermatogenesis.