Bone marrow-derived macrophages (BMDMs) are a key model system for studying macrophage biology in vitro. Commonly used methods to differentiate macrophages from bone marrow are treatment with either recombinant M-CSF or the supernatant of L929 cells, which secrete M-CSF. However, little is known about the composition of L929 conditioned media (CM) and how it affects BMDM phenotype. Here, we used quantitative mass spectrometry to characterise the kinetics of protein secretion from L929 cells over a two-week period. While M-CSF is very abundant in L929 CM, we identified several other immune-regulatory proteins at surprisingly high abundance. L929 CM induced a slightly pre-activated phenotype and the expression of a number of known innate immune proteins in macrophages. This resource will be valuable to all researchers using L929 CM for the differentiation of BMDMs.