Macrophage differentiation and polarization occur in vivo under the influence of the localized cytokine milieu. In vitro studies frequently rely on cellular differentiation in culture; hence, unrecognized variables could have a large influence on the observed cellular phenotype. We measured macrophage in vitro responses to fungal ligands (Aspergillus germ tubes and zymosan), focusing on the degree to which culture conditions impact stimulatory responses through the C-type lectin receptor, dectin-1, which is involved in both MyD88-dependent and MyD88-independent signaling in response to fungal beta1,3 glucan. Results show that macrophages harvested from different murine anatomic sites exhibit varying degrees of MyD88-dependence, with bone marrow-derived macrophages (BMDM) cultured in L929 conditioned medium (L929 CM) exhibiting the largest degree of MyD88-independence. After differentiation in recombinant MCSF (rMCSF), MyD88(-/-) macrophages have decreased surface expression of dectin-1 compared to wild type macrophages; however, culture in L929CM results in higher, and equivalent expression of dectin-1 on both MyD88(-/-) and wild type BMDM. In addition to MCSF, L929CM contains high amounts of VEGF, MCP-1, KC, and MIG, and low amounts of FGF-beta, Eotaxin, IL-10, IL-9, and IL-12. Thus, methods of in vitro maturation dictate variable inflammatory responses by MyD88(-/-) macrophages in association with altered expression of cell surface dectin-1. L929 conditioned medium is a suboptimal alternative to rMCSF for in vitro studies. As MyD88(-/-) BMDM exhibit low surface expression of dectin-1 after in vitro culture in rMCSF, differences in dectin-1 dependent, MyD88-independent signaling may account for some of the phenotypes currently ascribed to MyD88-deficiency alone.