The present study was conducted to evaluate the influence of culture media, pH and temperature on the growth dynamics of two strains of Flammulina velutipes (DMRX-166 and DMRO-253) for enhancing mycelium proliferation under in vitro conditions. Five different solid media were tested, and distinct differences in growth were observed between the strains and across the media. Among the media, Malt Extract Agar (MEA) supported the highest mycelial proliferation with average growth rate (5.45mm/day for DMRX-166 and 5.12 mm/day for DMRO-253), followed by Potato Dextrose Agar (PDA) and Wheat Straw Agar (WSDA). Irrespective of strain, among the four differenttemperature regimes (10°C, 15°C, 20°C and 25°C), the highest radial growth was recorded at 25 °C, while lower temperatures (10-15 °C) resulted in slower mycelial growth. Similarly, across pH levels (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0), optimal mycelial development occurred within the pH range of 7.0-8.0, with DMRX-166 showing maximal mycelial growth compared to DMRO-253. Maintaining consistent environmental conditions throughout the experiment was challenging. Nevertheless, the findings contribute valuable insights into the optimization of cultural conditions for efficient mycelial proliferation of F. velutipes, providing a scientific basis for large scale cultivation and strain improvement.