Milk thistle (Silybum marianum (L.) Gaertn) belonging to the Asteraceae family and known for its valuable secondary metabolite, silymarin. In order to get the Cell suspension culture of Silybum marianum, the combination of 2,4-D and BAP hormones each with five different concentrations (0, 0.1, 1, 2 and 5 mg/L) and 3 different seedling explants (cotyledon, hypocotyl and root) was used to callus formation. High-quality callus was observed in eight different of hormone-explant combinations. Successful Cell suspension culture was achieved only by using callus created from cotyledon explants treated with 0.1 mg/L 2,4-D and 5 mg/L BAP. Furthermore, the study examined the effects of 2% and 4% Serendipita indica Cell wall extract as an elicitor on silymarin production in Cell suspension culture at three different inoculation times (24, 48, and 72 h). After 48 h of inoculation with 2% fungal extract, the highest level of silymarin (199 ppm) was observed, which was significantly different from the control (46 ppm). The silymarin content of Cells increased over time through elicitation with 4% fungal elicitor, while no similar pattern was observed with 2% fungal extract. Based on the results, the Cell wall of S. indica can significantly enhance the amount of SLM in the Cell suspension culture of S. marianum.