Cytokines are key factors affecting the fate of intestinal stem cells (ISCs) and effective reagents to manipulate ISCs for research purpose. Tumor necrosis factor alpha (TNFα) is a cytokine produced primarily by monocytes and macrophages. It can induce apoptotic cell death and inflammation, and to inhibit tumorigenesis and viral replication. Additionally, TNFα has been shown to play a critical role in the pathogenesis of inflammatory bowel disease (IBD). It is therefore important to identify the mechanism by which individual cytokines affect particular cell types. For this purpose, we used both conventional (CONV) and altered Schaedler flora (ASF) C3H/HeN mice to elucidate the effect of different microbial populations (complex versus defined) on growth of miniguts derived from two different intestinal environments. Furthermore, we studied the effects of different concentrations of TNFα extracted from the lymph and spleen on the growth and viability of ISCs recovered from mice bearing the ASF or CONV microbiota. The effect of TNFα on miniguts growth depends not only on the source and concentration, but also on the intestinal microenvironment from which the ISCs were derived. The findings suggest that TNFα influenced the proliferation of miniguts derived from ISCs and, therefore, modulates mucosal homeostasis of the host.Copyright © 2020. Published by Elsevier B.V.