We used an ovalbumin (OVA)-specific αβ-TCR transgenic mouse system to examine the cellular basis of CD4+T helper (Th) phenotype development in vitro. Heat-killed Listerla monocytogenes (HKLM) strongly promotes the in vitro development of a Th1 phenotype in OVA-specific transgenic T cells. Listeria monocytogenes effects to promote the Th1 phenotype are antigen presenting cell (APC) dependent and occur when spienic APCs, but not the B cell hybridoma TA3, are present during T cell activation. However, addition of FACS-sorted macrophages to TA3 activated cultures restores the ability of Listeria to induce Th1 development. This effect on T cell development does not require MHC-restricted antigen presentation by macrophages, but may act through soluble factors. Although the presence of interferon γ is necessary for Listeria induction of Th1 development, IFN-γ alone is insufficient to induce Th1 development. Furthermore, Listeria induction of the Th1 phenotype does not require several known products of activated macrophages, including interleukin-1 (IL-1), tumor necrosis factor (TNF-α), IL-6, or nitric oxide. Although transforming growth factor-β (TGF-β) may mediate some Listeria effects, it does not fully reconstitute Listerla effects to promote Th1 development. In summary, host interactions with bacterial pathogens can affect the development of specific Th subsets, allowing Innate Immune cells to direct development of specific Immune phenotype. For Listeria monocytogenes, the induction of the Th1 phenotype may involve a novel cytokine distinct from several known factors produced by activated macrophages.