Impaired differentiation of RhoH-null DN3 and DN4 cells in vitro. (A) Sorted DN3 cells were cultured for 4 days on OP9-DL1 cells in the presence of IL-7 and Flt3 ligand. Differentiation from DN3 to DN4 was tested by FACS analysis of lineage-negative cells for the expression of CD44 and CD25. RhoH-null cells showed a significantly higher percentage of DN3 and a lower percentage of DN4. FACS staining for CD4 and CD8 revealed significantly increased levels of DN and reduced levels of DP in the absence of RhoH. Furthermore, cellularity of the RhoH-null cultures was approximately 5-fold decreased, indicating defective expansion in the absence of RhoH. **P < .01; ***P < .001. Error bars show the standard deviation (n [control]/[RhoH^−/−]: 2/3). (B) Sorted DN4 cells were cultured for 4 days on OP9-DL1 cells in the presence of IL-7 and Flt3 ligand. FACS staining for CD4 and CD8 revealed significantly increased levels of DN and reduced levels of DP in the absence of RhoH. Furthermore, cellularity of the RhoH-null cultures was approximately15-fold decreased, indicating defective expansion in the absence of RhoH. **P < .01. Error bars show the standard deviation (n [control]/RhoH^−/−]: 3/4.