(A) Schematic explaining genetics used to delete En1/2 in the eCN and/or GCPs. Atoh1-tTA; tetO-Cre; En1lox/lox; En2lox/lox animals were treated with Dox from either E13.5 until sacrifice to specifically knockout En1/2 in eCN (eCN-En1/2 CKO) or between E8.5 and E12.5 to knockout En1/2 in GCPs (GCP-En1/2 CKO). No Dox was given to knockout En1/2 in both cell types in the cerebellum (eCN+GCP-En1/2 CKO). (B) Staining for lacZ activity on sections of Atoh1-tTA; tetO-Cre; R26LSL-LacZ/+ animals at P7 showing the specificity of the Dox regimens. (C) Quantification of cerebellum area in the vermis showing reduction only in eCN+GCP- and eCN-En1/2 CKOs compared to their littermate controls at P30 (n = 4 animals/condition, Two-way ANOVA, F(1,24)=8.042, p=0.009). (D,E) Quantification of cerebellum area based on location showing the area is only reduced in the vermis and paravermis, but not in the hemispheres at P30 in eCN+GCP-En1/2 CKOs (D, n = 4 animals/condition Two-way ANOVA, F(1,6)=12.88, p=0.011) and eCN-En1/2 CKOs (E, n = 6 animals/condition, Two-way ANOVA, F(1,10)=8.34, p=0.016, *Student’s t-test in the paravermis). (F–S) Quantifications by sector in the vermis for: overall area (F,M), for IGL (G,N) or ML (H, O) area relative to total sector area, for PC numbers (I,P) and for density of PCs (J,Q), or ML interneurons (K,R) or GCs (L, S) in eCN+GCP-En1/2 CKOs and eCN-En1/2 CKOs. A reduction in the ASec area is seen in both mutants (eCN+GCP-En1/2 CKO: Two-way ANOVA, F(1,18)=22.07, p=0.0002, eCN-En1/2 CKOs: Two-way ANOVA, F(1,36)=12.4, p=0.0012) and PC numbers in the ASec (eCN+GCP-En1/2 CKO: Two-way ANOVA, F(1,18)=32.51, p<0.0001, eCN-En1/2 CKOs: Two-way ANOVA, F(1,36)=13.5, p=0.0008), however, cell densities are preserved. Significant post hoc comparisons are shown in the figure.