The striatum, a critical component of the basal ganglia, is essential for motor control, cognitive processing, and emotional regulation. Medium spiny neurons (MSNs) are the primary neuronal population in the striatum, classified into D1 and D2 subtypes. The transcription factor Pou3f1 has been hypothesized to play a crucial role in the development of pyramidal neurons. Recently, a comprehensive analysis of the human embryonic scRNA-seq dataset predicted and emphasized the bridging function of POU3F1 between striatal progenitor cells and immature neurons, though this finding lacked genetic validation. In this study, we found that Pou3f1 expression was significantly reduced after Six3 deletion. However, Pou3f1 deletion does not significantly affect the number or subtype composition of MSNs, nor the proliferation and differentiation of progenitor cells, in our Pou3f1 conditional knockout (cko) mice, challenging the in silico predictions based on human data. These results suggest that Pou3f1 is not required for the specification, generation, or differentiation of MSNs, though its potential involvement in other aspects of striatal development cannot be entirely ruled out.