X-VIVO 15 with Gentamicin and Phenol Red, 1 L is a fully chemically defined, serum-free hematopoietic cell culture medium. It is used for investigating second messenger systems in the activation of human and murine lymphocytes and provides a nutritionally balanced medium for selected hematopoietic cell culture applications. The medium is described for use with tumor-infiltrating lymphocytes, lymphokine-activated killer cells, and peripheral blood lymphocytes. Its defined composition is free of exogenous growth factors, artificial stimulators such as protein kinase C, and undefined supplements, supporting controlled culture conditions where these components should be avoided. This X-VIVO 15 formulation contains gentamicin and phenol red and is completed with pharmaceutical-grade human albumin, recombinant human insulin, and pasteurized human transferrin. The 1 L format offers a larger volume for workflows using this serum-free hematopoietic cell culture medium and the listed formulation components in defined hematopoietic cell culture conditions.