The pathogenic differences between CXCR4 (X4)- and CCR5 (R5)-utilizing strains of HIV-1 may be predominantly due to differences in viral tropism, which in turn may be due to differential coreceptor utilization. We tested this hypothesis in the human thymus organ of the SCID-hu Thy/Liv mouse, using recombinants of NL4-3 that were isogenic except for Env coreceptor-binding determinants of the V1-V3 loops. Conversion of NL4-3 from an X4 to an R5 isolate was associated with altered tropism for cell subpopulations within the Thy/Liv organ (with a higher frequency of infection of thymic stromal cells, including macrophages), a slower rate of replication, and a lower level of cytopathicity. These observations underscore the causal relationships between tropism, coreceptor use, and cytopathicity in the human thymus in vivo.