Gut CD4 T cells are major targets of HIV-1 and are massively depleted early during infection. To better understand the mechanisms governing HIV-1-mediated CD4 T cell death, we developed the physiologically-relevant Lamina Propria Aggregate Culture (LPAC) model. The LPAC model is ideal for studying CD4 T cell death induced by clinically-relevant Transmitted/Founder (TF) HIV-1 strains and is also suitable for studying how enteric microbes and soluble factors (e.g., Type I Interferons) impact LP CD4 T cell death and function. Here, we detail the protocol to establish LP CD4 T cell infection using a process of spinoculation, the subsequent evaluation of infection levels using multicolor flow cytometry and the determination of overall LP CD4 T cell death using absolute LP CD4 T cell counts. We also describe the preparation of virus stocks of Transmitted/Founder (TF) HIV-1 infectious molecular clones that were successfully used in the LPAC model.