Sequoia sempervirens (D. Don) Endl. are relict giant plants whose reproductive vulnerability cannot be challenged. Currently, the depletion of natural populations and the risk of plant extinction are acute issues. Therefore, biotechnological methods for creating in vitro genetic banks and bioresource collections are becoming particularly relevant. Such technologies include clonal micropropagation, which allows obtaining genetically stable, “healthy” planting material with a high reproduction rate. Extractive substances from Sequoia sempervirens tissues exhibit high biological activity. Aseptic in vitro cultures also retain the ability to biosynthesize substances with high biological activity. It is known that in vitro gymnosperms cultures are challenging to initiate and exhibit slow growth rates. Therefore, the study aimed at overcoming these methodological barriers is of particular importance. This study presents a method for obtaining callus tissue from the loewr apex of S. sempervirens microclones cultivated on Murashige and Skoog (MS) nutrient medium supplemented with various auxins (2,4-D, IAA, IBA, and NAA) at a concentration of 3 mg/L. The nutrient medium variant with NAA showed the earliest activation of the dedifferentiation process. In contrast, callus cultivated on the medium with IAA exhibited a low growth rate and developed a dark brown colouration.