Ribosome biogenesis is intricately linked to the pathogenesis of B-cell acute lymphoblastic leukemia (B-ALL), but the precise regulatory mechanisms remain to be elucidated. First of all, our study identify that RPS15a, the ribosomal small subunit structural protein, is significantly higher expressed in B-ALL patients, indicating a potential crucial role of RPS15a in modulating B-ALL progression. Next in importance, our findings found that RPS15a knockdown (KD) results in the impede of cell proliferation and cell cycle progression, while concurrently inducing apoptosis of B-ALL cells. Next, we used immunofluorescence staining of nucleolar proteins NPM1 and FBL to evaluate whether RPS15a KD-mediated abnormal nucleolar ribosome assembly triggers nucleolar stress. Our findings revealed that, in comparison to control B-ALL cells, RPS15a KD disrupted the localization of NPM1 and FBL within the nucleus, altered the normal architecture of the nucleolus, and induced nucleolar stress. Mechanistically, our study found that RPS15a KD impairs the ribosomal biogenesis by suppressing the synthesis and processing of ribosomal RNAs (rRNAs), including pre-rRNA, 18S rRNA, 28S rRNA, and 5.8S rRNA. Then, we observed that the depletion of RPS15a leads to the activation of p53 signaling, accompanied by an increase in the protein expression of p53 and p21, which are critical signals that induce nucleolar stress. Furthermore, our rescue experiment involving p53 KD found that the suppression of p53, following RPS15a KD, can alleviate ribosomal biogenesis, facilitate cell cycle progression and inhibit apoptosis. In conclusion, these findings suggest that RPS15a primarily regulates the progression of B-ALL by modulating p53 signaling-mediated nucleolar stress, thereby identifying RPS15a as a critical target for B-ALL treatment.